Derivatives of branched-chain lipophilic molecules and uses thereof

ABSTRACT

The present invention discloses novel phospho-derivatives of branched-chain lipophilic molecules useful for permeabilizing biological barriers and for inhibiting tumor growth. The invention further discloses pharmaceutical compositions comprising said molecules and their uses.

FIELD OF THE INVENTION

[0001] The present invention relates to novel phospho-derivatives ofbranched-chain lipophilic molecules, to pharmaceutical compositionsthereof and to use thereof for increasing permeability of biologicalbarriers in a reversible and selective manner and for inhibiting tumorgrowth.

BACKGROUND OF THE INVENTION

[0002] A major limitation in the use of many drugs and therapeuticagents is their inadequate ability to pass through biological barriers.This presents a serious problem especially for the treatment of diseasesand disorders in privileged sites such as the central nervous system(CNS).

[0003] The blood brain barrier (BBB), made up of specializedmicrovascular endothelial cells connected by tight junctions, isnormally responsible for maintaining the homeostatic environment of thebrain and protecting it from toxic agents and degradation productspresent in the circulatory system. However, in certain pathologicalsituations, the presence of the BBB may interfere with the transport oftherapeutic substances into the brain, thus hampering treatment ofcentral nervous system lesions, including tumors, infections, abscessesand degenerative diseases.

[0004] In a similar way, the presence of the blood tumor barrier (BTB)interferes with the delivery of chemotherapeutic agents into the tumor,thus decreasing drug bioavailability and preventing efficienttherapeutic effect where it is needed. The problem of insufficientaccess of the therapeutic agent to the diseased target is especiallysevere in the case of CNS tumors, and patients bearing malignant braintumors have poor prognoses.

[0005] In order to achieve clinically useful concentrations of certaindrugs at restricted sites, it is often required to administer thesecompounds at high systemic dosages. The high systemic concentrations, inturn, are associated with adverse side effects and high levels oftoxicity.

[0006] One strategy for attacking the problem involves altering thebiophysical characteristics of hydrophilic drug molecules, for example,by linking these drugs to a lipophilic carrier. Since drug permeabilityacross such biological membranes depends on its lipophilicity,increasing the lipophilic nature of the compound should, theoretically,improve its bioavailability and increase therapeutic effects. Suchcovalent polar lipid conjugates with neurologically active compounds fortargeting are disclosed in U.S. Pat. No. 5,827,819 to Yatvin et al.

[0007] Another approach to circumvent the BBB impermeability is byemploying agents that transiently open the BBB and facilitate the entryof a particular drug or agent into the brain. Agents such as mannitolhave been shown to exert this desirable effect and have been employed inthe delivery of chemotherapeutic agents to malignant brain tumors(Hiesinger et al. (1986), Annals of Neurology, 19:50-59). The use ofthis kind of hyperosmolar BBB disruption in brain tumor therapy has,however, been controversial since, in addition to the drug crossing theBBB, other molecules such as neurotoxins are also permitted entry. Thismay account for the high incidence of stroke, seizures, immunologicalreactions and ocular toxicity associated with treatment using osmoticopening methods.

[0008] A variety of other treatments have also been disclosed thatincrease permeability of the blood brain barrier including: the use ofbradykinin agonists (WO 91/16355 of Alkermes) and certain other peptides(WO 92/18529 of Alkermes); use of bacterial cell wall fragments (WO91/16064 of the Rockefeller Univ.) or the use of antibody to Bordetellapertussis filamentous haemagglutinin or brain endothelial x-molecule (WO92/19269 of the Rockefeller Univ.). Certain fatty acids such as oleicacid have also been reported to reversibly open the BBB (Sztriha andBetz (1991), Brain Res. 336: 257-262).

[0009] The usefulness of methods for reversibly increasing thepermeability of the blood brain barrier prior to administration ofdiagnostic reagents (U.S. Pat. No. 5,059,415 of the Oregon Health Sci.U.) or therapeutic reagents (WO 89/11299 of the Oregon Health Sci. U.)have been disclosed.

[0010] It was previously disclosed by the inventors of the presentinvention, in International patent application publication number WO99/02120, that branched fatty acids and certain lipophilic derivativesthereof are useful for reversibly permeabilizing biomembranes. However,compounds which comprise a phosphate moiety have not been disclosed.Furthermore, it has not been disclosed that by modifying the branchedfatty acids by addition of a phosphate moiety it is possible to modulatethe opening of biological barriers in a specific and differentialmanner.

[0011] Clearly, the compositions developed so far for permeabilizingbiological membranes and barriers produce severe side-effects.Therefore, there is an unmet need for providing effective and safe meansfor delivering adequate quantities of therapeutic and diagnostic agentsinto restricted sites.

[0012] Numerous compositions have been proposed for use in treatingvarious cancers, included among them are compounds comprising ahydrocarbon chain and a phosphocholine moiety. U.S. Pat. Nos. 4,837,023and 5,049,552, both to Eibl, disclose compositions and methods useful intreating cancer. The active material in these cases is the knownsubstance Hexadecylphosphocholine (HePC). However, according to thedisclosure in those patents, of all compounds tested only HePC possesseda practically useful anti-tumor action, while homologues with shorteralkyl radicals possessed no or much too low anti-tumor action andhomologues with longer alkyl radicals were much too toxic. The prior artneither discloses nor suggests any of the compounds which are thesubject matter of the present invention.

SUMMARY OF THE INVENTION

[0013] The present invention provides, in one aspect, a compound of thegeneral formula I:

[0014] or a pharmaceutically acceptable salt thereof, wherein:

[0015] R1 and R2 are the same or different, saturated or unsaturatedaliphatic chain comprising from 2 to 30 carbon atoms;

[0016] R3 is A-[CH₂]_(m)-B-[CH₂]_(n)-C-[CH₂]_(p)-D wherein m, n and pare each independently zero or an integer from 1 to 12, and A, B, C andD are each independently selected from a covalent bond, amino, amido,oxygen, thio, carbonyl, carboxyl, oxycarbonyl, thiocarbonyl, phosphate,amino phosphate mono- di- and tri-amino phosphate group with the provisothat no two oxygen atoms are directly connected to each other;

[0017] Z₁ and Z₂ are the same or different, each may be absent orindependently selected from a) hydrogen, sodium, lithium, potassium,ammonium, mono-, di-, tri- and tetra-alkylammonium, or b) together withthe phospho group form a phospho ester of glycerol, choline,ethanolamine, inositol, serine, mono- or oligosaccharide.

[0018] In one preferred embodiment, the compound of the general formulaI is an α-branched fatty molecule wherein R1 and R2 are hydrocarbonchains having, respectively, 3 and from 12 to 16 carbon atoms.

[0019] According to another preferred embodiment, R3 of the compound ofthe general formula I comprises mono- or di-ethylene glycol moiety.

[0020] Currently preferred compounds according to the invention are:

[0021] 4-Hexadecyl phosphate (3,12-PO₄),

[0022] 4-Octadecyl phosphate (3,14-PO₄),

[0023] 4-Eicosanyl phosphate (3,16-PO₄),

[0024] 8-Pentadecyl phosphate (7,7-PO₄),

[0025] 4-Hexadecanoyloxyethyl phosphate (3,12-MEG-PO₄),

[0026] 2-(4′-Hexadecanoyloxy)ethoxyethyl phosphate (3,12-DEG-PO₄),

[0027] 4-Hexadecanyloxyethyl phosphate (3,12-(ether)-MEG-PO₄),

[0028] 2-(4′-Hexadecanyloxy)ethoxyethyl phosphate(3,12-(ether)-DEG-PO₄),

[0029] 4-Hexadecyl phosphocholine (3,12-PC),

[0030] 2-(4-Hexadecanoyloxy)ethyl phosphocholine (3,12-MEG-PC),

[0031] 2-(4-Hexadecanoyloxy)ethoxyethyl phosphocholine (3,12-DEG-PC),

[0032] 2-{2′-[10″-(Hexadecyl-4-oxy)decyl-1-oxy]ethoxy}ethylphosphate(3,12-O-C₁₀-DEG-PO₄),

[0033]2-{2′-[10″-(Hexadecyl-4-oxy)decyl-1-oxy]ethoxy}ethylphosphocholine(3,12-O-C₁₀-DEG-PC),

[0034] 4-Octadecanoyloxyethyl phosphate (3,14-MEG-PO₄),

[0035] 2-(4′-Octadecanoyloxy)ethoxyethyl phosphate (3,14-DEG-PO₄),

[0036] 4-Octadecanyloxyethyl phosphate (3,14-(ether)-MEG-PO₄),

[0037] 2-(4′-Octadecanyloxy)ethoxyethyl phosphate(3,14-(ether)-DEG-PO₄),

[0038] 4-Octadecyl phosphocholine (3,14-PC),

[0039] 2-(4-Octadecanoyloxy)ethyl phosphocholine (3,14-MEG-PC),

[0040] 2-(4-Octadecanoyloxy)ethoxyethyl phosphocholine (3,4-DEG-PC),

[0041] 4-Eicosanoyloxyethyl phosphate (3,16-MEG-PO₄),

[0042] 2-(4′-Eicosanoyloxy)ethoxyethyl phosphate (3,16-DEG-PO₄),

[0043] 4-Eicosanyloxyethyl phosphate (3,16-(ether)-MEG-PO₄),

[0044] 2-(4′-Eicosanyloxy)ethoxyethyl phosphate (3,16-(ether)-DEG-PO₄),

[0045] 4-Eicosanyl phosphocholine (3,16-PC),

[0046] 2-(4-Eicosanoyloxy)ethyl phosphocholine (3,16-MEG-PC),

[0047] 2-(4-Eicosanoyloxy)ethoxyethyl phosphocholine (3,16-DEG-PC),

[0048] 10-(4′-Hexadecanoyloxy)decanyl phosphate,

[0049] 10-(8′-Pentadecanoyloxy)decanyl phosphate,

[0050] 2-[2′-(2″-Propyleicosanoyloxy)-ethoxy]ethyl Phosphate(3,18-DEG-PO₄), and

[0051] 2-(2′-Propyleicosanoyloxy)ethoxy ethylphosphocholine(3,18-DEG-PC).

[0052] Currently most preferred compounds are:

[0053] 2-(4′-Hexadecanoyloxy)ethoxyethyl phosphate, monosodium salt,

[0054] 2-(4′-Hexadecanoyloxy)ethoxyethyl phosphate, diosodium salt,

[0055] 2-(4′-Hexadecanyloxy)ethoxyethyl phosphate,

[0056] 2-(4-Hexadecanoyloxy)ethyl phosphocholine, and

[0057] 2-(4-Hexadecanoyloxy)ethoxyethyl phosphocholine.

[0058] Compounds of the invention are useful for increasing permeabilityof biological barriers. Thus, in another aspect, the invention providespharmaceutical compositions comprising an effective amount of a compoundof the general formula I depicted above, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.

[0059] The pharmaceutical compositions of the invention may furthercomprise a pharmaceutically effective amount of a biologically activeagent.

[0060] In one preferred embodiment, the biologically active agent is atherapeutic agent. The therapeutic agent may be selected from, but isnot limited to, anti-tumor, anti-viral anti-microbial, anti-fungal,anti-inflammatory, neuroprotective agents and bioactive peptides andproteins.

[0061] In another preferred embodiment, the pharmaceutical compositionsof the invention further comprise a diagnostic agent. The pharmaceuticalcompositions are useful for facilitating administration of biologicallyactive molecules, for example therapeutic and diagnostic agents, intotissues and organs, in particular at privileged sites which areprotected by biological barriers.

[0062] In one particular embodiment, the pharmaceutical compositions areuseful for increasing drug delivery across the blood retinal barrier(BRB), blood brain barrier (BBB) and blood tumor barrier (BTB). Thepharmaceutical compositions, in accordance with the invention, may alsobe useful for increasing permeability of other biological barriers,thus, for example, facilitating absorption through the ski, cornea,conjunctival, nasal, bronchial, buccal, vaginal and the gastrointestinalepithelium, and across the blood testis barrier and blood kidneyinterphase.

[0063] The pharmaceutical compositions may be administered by oral,parenteral or topical administration or by regional perfusion, enema orintra-organ lavage. Preferably the pharmaceutical compositions of theinvention are intra-arterially or intra-thecally administered.

[0064] In yet another aspect, the present invention provides methods forincreasing permeability of biological barriers. These methods compriseexposing said barriers to an effective amount of a compound of thegeneral formula I, or pharmaceutically acceptable salt thereof, thusenabling or increasing permeability of the biological barrier.

[0065] In still another aspect, the present invention provides a methodfor administration of a biologically active agent into a privileged siteor organ comprising exposing said site or organ to said biologicallyactive agent in the presence of an effective amount of a compound inaccordance with the invention, thus enabling or increasing thepenetration and/or accumulation of the biologically active agent in theprivileged site or organ.

[0066] The privileged site or organ may be selected from, but is notlimited to, the spinal cord, brain, eye, testis, glands and tumors.

[0067] In still another aspect, the present invention provides a methodfor treatment of a tumor comprising administering to a patient in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising as an active ingredient a compound of the generalformula I in accordance with the invention. Said tumor may be selectedfrom, but not limited to, carcinoma (e.g. breast, colon, rectal andbladder carcinomas), glioma (e.g. astrocytoma), neuroblastoma,retinoblastoma, intraocular malignancy, lymphoma, leukemia, sarcoma andmelanoma. The tumor may be a primary or secondary tumor.

[0068] The invention further provides a method for treatment of acentral nervous system disease or disorder comprising administering to apatient in need thereof a therapeutically effective amount of apharmaceutical composition comprising a compound of the general formulaI, or a pharmaceutically acceptable salt thereof, in combination with atherapeutic agent. The therapeutic agent may be included in the samepharmaceutical composition comprising the compound of the generalformula 1, or in a separate composition.

[0069] In a preferred embodiment the treated disease in the centralnervous system is a brain tumor and the therapeutic agent is ananti-cancer drug. In another preferred embodiment the treated disease isan ophthalmologic disease or disorder, for example, cystoids macularedema (CME), Age-related macular degeneration (ARMD), intraocularinfections, intraocular inflammations and intraocular malignancies.

[0070] In still a further aspect, the present invention provides amethod for increasing accumulation of a diagnostic agent in an organprotected by a biological barrier comprising administering to anindividual said diagnostic agent in combination with an effective amountof a compound of the general formula I as defied above, thus increasingaccumulation of the diagnostic agent in the organ protected by abiological barrier. The diagnostic agent may be included in the samepharmaceutical composition comprising the compound of the generalformula I, or in a separate composition.

[0071] In one preferred embodiment, said organ protected by a biologicalbarrier is the central nervous system.

BRIEF DESCRIPTION OF THE DRAWINGS

[0072]FIG. 1 depicts a graph correlating the various chain lengths ofα-branched fatty acids of the 3,n-type, with the potency of thesecompounds in extravasation of Evans blue-albumin complex into rat brain.

[0073] FIGS. 2A-C depict Evans blue accumulation in brain sections ofrats bearing bilateral gliomas following unilateral administration of acompound as follows: 10 μM 3,12-DEG-HPO₄Na (FIG. 2A), 40 μM 3,12-Na(FIG. 2B) or mannitol 25% (FIG. 2C).

[0074] FIGS. 3A-C depict fluorescein sodium salt (F—Na) angiography ofthe posterior part of a rat eye as recorded at 30 seconds and 10 minutesfollowing administration of F—Na either alone (FIG. 3A), or incombination with 3,12-DEG-HPO₄Na (FIG. 3B) or vehicle (FIG. 3C).

[0075] FIGS. 4A-B depict F—Na accumulation at the anterior part of theeye as recorded 10 minutes after F—Na administration accompanied witheither 3,12-DEG-HPO₄Na (FIG. 4A) or vehicle (FIG. 4B) following theprocedure described in Example 26.

DETAILED DESCRIPTION OF THE INVENTION

[0076] The present invention relates to compounds, compositions andmethods for increasing permeability of biological barriers and forinhibiting tumor growth.

[0077] A desirable agent for facilitating delivery of therapeutic ordiagnostic agents into restricted sites (e.g. the brain, eye, testisetc.) is one that is capable of permeabilizing the limiting biologicalbarrier. However, preferred permeabilization has to be accomplished in adifferential fashion without producing unacceptable degree of sideeffects. For example, in the case of treating a brain tumor, it would beadvantageous to use an agent that permeabilizes the BTB to a muchgreater extent than the BBB in the intact neighbouring brain tissue.Specific permeabilization, in this case, enables preferentialaccumulation of toxic drugs in the pathological tissue while causingminimal or no damage to the surrounding healthy brain tissue. Similarconsiderations apply for other biological barriers. For example, it isdesirable to have an agent that is capable of increasing delivery ofdrugs or other beneficial molecules across skin and intestine barriersin a transient and specific fashion.

[0078] The present invention concerns novel compounds and is based onthe unexpected finding that these compounds may increase permeability ofbiological barriers-in a reversible and selective manner. According tothe teaching of the present invention, the novel compounds are of thegeneral formula I as defined hereinabove or pharmaceutically acceptablesalts thereof.

[0079] In the specification, the compounds of the general formula I willbe collectively referred to as “DP-BFAs” or interchangeably by thegeneral name phospho-derivatives of branched chain lipophilic moleculesand in short “P-BFA”. Branched chain lipophilic molecules not bearing aphosphate moiety will be referred to as “BFA” or “carbo-BFA”.

[0080] In one preferred embodiment the P-BFAs are phospho-derivatives ofα-branched lipophilic molecules of the general structure R1(R2)—CH—. Inother embodiments according to the present invention, the P-BFAs arephospho-derivatives of branched lipophilic molecules of the generalstructure R1(R2)—CH—[CH₂]_(m)— wherein m is from 1 to 12, thus referringto ω-branched lipophilic molecules branched, for example, at position β(m=1), γ (m=2) etc.

[0081] Specific DP-BFAs will be referred to, hereinafter, by theirparticular R1 and R2 that represent, respectively, the number of carbonatoms on the side- and main-chain of the branched chain lipophilicmolecule.

[0082] Compounds of the general formula I, wherein R3 ismono-ethyleneglycol or di-ethyleneglycol will be referred to asR1,R2-MEG-PO₄ and R1,R2-DEG-PO₄, respectively. In some cases, the bondlinking the branched chain moiety and the adjacent chemical group isspecifically indicated as, for example, in 3,12-O-C₁₀-DEG-PO₄,3,12-(ether)-DEG-PO₄ etc. Compounds of the general formula I wherein Z₁is choline will be referred to as R1,R2-PC.

[0083] In accordance with the teaching of the present invention, it ispossible, by varying the various components in the structure of thecompounds of the general formula I, to achieve fine-tuning in stabilityof the molecules and their permeabilization effect on biologicalbarriers. For example, varying the number of the carbon atoms in thealkyl groups R1 and R2 and the level of saturation affect the overallhydrophobicity of the molecule, thus enabling opimization of its abilityto cross specific biological membranes.

[0084] Currently preferred compounds in accordance with the inventionare DP-BFA molecules wherein the length of the carbon chain in R1 isfrom 2 to 14, and the total number of carbon atoms in R2 and R3 togetheris from 6 to 26. Currently more preferred compounds are α-branched chaincompounds wherein R1 is 3 carbon atoms and R2 is from 12 to 16 carbonatoms. Preferred compounds of the general formula I comprise analiphatic chain having up to 20 carbon atoms in length counted from thephosphate group to the branching point of the branched lipophilicmolecule. This estimated number of carbon atoms in the R3 moiety of thegeneral formula I as defined above, is most suitable for effectivemembrane perturbation by the DP-BFA molecule and thus for eliciting ofthe desirable permeabilization effect. It should be noted that theabove-mentioned aliphatic chain may be a continuos hydrocarbon chain, ormay be interrupted by one or more hetroatoms selected from the group ofoxygen, sulphur, nitrogen and phosphorus atoms, as included in thedefinition of R3.

[0085] In one preferred embodiment in accordance with the invention, R3of the molecule of the general formula I includes a carbonyl grouplinking the branched chain moiety and at least one glycol moiety via anester bond. In another preferred embodiment, R3 includes a covalent bondlinking the branched chain moiety and at least one glycol moiety via anether bond. Under physiological conditions, ether bonds are generallyless susceptible to enzymatic cleavage, therefore are expected to bemore stable than ester bonds.

[0086] Another factor that may affect the ability of DP-BFA compounds topermeabilize various biological barriers is the polarity of thedifferent chemical groups in the molecule. This also may contribute tothe fine tuning of the differential permeabilization effect.

[0087] Preferred compounds of the invention are in salt forms, beingeither mono- or di-salt compounds of the general formula I. Suitablesalts may include any pharmaceutically acceptable salt comprising amonovalent or divalent counter ion which may be selected from, but isnot limited to, Na⁺, Li⁺, K⁺, NH⁴+, Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, mono-, di-, tri-and tetra-alkylammonium. More preferred compounds comprise a monovalentsalt. Particularly preferred are DP-BFA molecules in the sodium saltform, being either mono- or di-sodium salts.

[0088] The compounds of the invention may be prepared by chemicalsynthetic methods well known in the art. Some of these methods areillustrated hereinafter in the Examples. Alternative procedures known tothose skilled in the art may also be employed.

[0089] The compounds of the invention were found to be useful indisrupting tight cell junctions in vitro and in increasingpermeabilization of biological barriers in vivo. Furthermore, thepresent invention is based on the unexpected finding that certainderivatives of phospho-branched chain lipophilic molecules havedifferential effects on opening of the BBB and BTB barriers.Accordingly, the various P-BFA compounds of the invention may be usefulin exerting permeabilization of biological barriers while producingminimal toxicity and side effects.

[0090] Some DP-BFA compounds are selective in terms of being capable todifferentially permeabilizing biological barriers to molecules ofdifferent sizes. This characteristic may attribute to higher safetylevels of the phospho-BFA agents. Some DP-BFAs differ in the duration oftheir effect which may be proven as another beneficial factor in certaintherapeutic circumstances wherein transient selective permeabilizationof a limiting barrier is desirable.

[0091] The compounds of the invention and pharmaceutical compositionscomprising them may enable or facilitate delivery of biologically activeagents across biological barriers.

[0092] The term “biologically active agents” is intended to encompassall naturally occurring and synthetic compounds capable of eliciting abiological response, having an effect on biological systems or servingas indicative tools. The biologically active agents may be therapeuticor diagnostic agents.

[0093] Therapeutic agents may include, but are not limited to,anti-neoplastic, anti-proliferative, anti-inflammatory, neurological,antibacterial, anti-mycotic aid antiviral agents. These compounds incombination with the compounds of the invention are particularly usefulin the treatment of pathological conditions, diseases and disorders inrestricted sites. For example, in the treatment of central nervoussystem and lesions including tumors, infections, abscesses anddegenerative disorders.

[0094] Diagnostic agents may include, but are not limited to, imagingagents, contrast agents and dyes. Examples of diagnostic agents includeradioactively labelled substances (e.g. Technetium-99m and Fluoride-18based agents) and contrast agents such as gadolinium based compounds.The compounds of the invention may be useful, for example, in methodsfor diagnosing and characterizing brain lesions. In this case, acompound of the invention used for increasing BBB permeability, may beco-introduced (either in the same or separate composition) with achemical agent that is labelled in such a way that it can be monitored.

[0095] The chemical agent to be monitored may be, for example, a generalindicator to brain cells or structures, or one that binds or accumulatesspecifically and exclusively in certain brain areas or brain lesions.The brain, then, may be analyzed to determine the presence of labellingagent. Analysis may be performed using scanning and imaging means knownin the art (e.g. magnetic resonance imaging techniques (MRI), positronemission tomography (PET) and Computed Tomography (CT)).

[0096] In other embodiments of the invention, the DP-BFA compounds maybe useful for enhancement of drug absorption in the intestines orthrough the skin. The biological barriers in the intestines and skinprevent passive diffusion of a number of substances across thegastrointestinal or skin epithelium thus preventing effective absorptionof certain useful chemicals, nutrients and drugs. This obstacle may beovercome by applying the desirable useful chemicals, nutrients and drugsin combination with a DP-BFA compound.

[0097] It will be readily apparent to those of ordinary skill in the artthat a wide range of biologically active compounds are appropriate foruse in combination with the branched lipophilic molecules of the generalformula I, therefore are included within the scope of the invention ascompounds useful in pharmaceutical compositions or methods of treatmentor diagnosis in accordance with the invention. In accordance with theinvention, the P-BFA compound is to be administered in combination withthe desirable biologically active agent. It should be clarified that thedesirable biologically active agent may be included in the samepharmaceutical composition of the compound of the general formula I, ormay be administered in a separate composition. Preferably both activeagents, i.e. the compound of the general formula I and the-desirablebiologically active agent(s), are administered simultaneously or withina short period of time from one another. The biologically activeagent(s) may be administered prior or, subsequent to the administrationof the P-BFA compound, provided that it will be within a time windowsuch that the effect of the P-BFA on the biological barrier issufficient to facilitate the passage of the relevant agents into therestricted site.

[0098] It is also possible that the desirable biologically active agentis not administered around the same time of the P-BFA administration,but is already present in the blood (being either naturally generated inthe body or produced as a slow release drug). Some examples includebioactive peptides and proteins such as neurotransmitters, growthfactors; hormones, antibodies etc. In this case the requirement is thatthe permeabilization effect of the target barrier occurs whilesufficient amount of the desirable biologically active agent is presentto exert its biological effect.

[0099] The improved activity of the compounds of the invention inpermeabilization of biological barriers results in increasedbioavailability of administered drugs, thus extending the therapeuticsusefulness of these drugs to conditions that do not respond to lowerdoses of drugs. This is especially relevant in treatment of diseases anddisorders in restricted sites, e.g. brain and the eye.

[0100] In addition, the compounds of the invention are advantageousinasmuch as they enable decrease in the useful dosage of drugs andconsequently reduction in undesirable systemic side effects.Furthermore, since the molecules of the invention enable selectivepermeabilization of barriers, they may preferentially increase druguptake at a specific site (e.g. tumor) and not in the neighboring cellsand tissues.

[0101] While examining the permeabilization effect of the P-BFA in vivoin tumor-bearing rats, it was surprisingly found that some of the testedcompounds showed remarkable-cytotoxic effects on the tumor, demonstratedin their ability to significantly inhibit the growth of the treatedtumor. Thus, it was established that certain compounds of the inventionmay be useful as anti-cancer agents. Moreover, some of the compounds ofthe invention, have demonstrated differential cytotoxic activity whentested for their effect on various normal and tumur cells in vitro.

[0102] In accordance with the principles of the present invention, thevarious DP-BFA molecules may be specifically tailored to suit specifictarget sites and specific indications. For example, molecules of theinvention were found to act in a differential manner in opening the BBBand BTB barriers. In this case the permeabilization of the BTB bycertain DP-BFAs is to a greater extent comparing to the effect on theBBB. Another advantageous characteristics of the effect of some DP-BFAmolecules, is the preservation of certain levels of discrimination as tothe compounds permitted to cross. Thus the DP-BFAs affect barrierpermeabilization in a more selective manner comparing to carbo-BFAs orhyperosmotic agents, such as mannitol. As a result, the P-BFA compoundsare expected to cause less toxic side effect in comparison to otherpermeabilizers known in the art. Furthermore, as their permeabilizationeffect was found to be reversible, the compounds of the invention mayalso be useful for chronic administration of drugs.

[0103] As mentioned in the specification and claims, an “effectiveamount” of P-BFA refers to that amount of a compound of the inventionwhich exerts the desirable beneficial effect in accordance with theinvention. According to one aspect of the invention, it is the amount ofP-BFA that significantly increases the permeability of a relevantbarrier to a molecule of interest. Namely, the amount of P-BFA whichincreases the permeability of the relevant barrier to allow sufficientquantities of a molecule of interest to cross the biological barrier soto exert its therapeutic or prophylactic effect or allow diagnosticprocedures. According to another aspect of the invention, it is theamount of P-BFA that is therapeutically effective as anti-cancer agent.Namely, that amount of P-BFA which inhibits uncontrolled cell growth.

[0104] The effective amount will be determined on an individual basisand will be based, at least in part, on consideration of theindividual's size, the specific disease, the severity of the symptoms tobe treated, etc. Thus, the effective amount can be readily ascertainedby a person of skill in the art employing such factors and using no morethan routine experimentation.

[0105] The dose range and the regimen employed will be dependent on theroute of administration, the age, sex, health and weight of therecipient and on the potency of the particular DP-BFA and the relevantuseful drug or agent administered. The skilled artisan will be able toadjust the DP-BFA compositions and dosage in order to obtain the desiredduration and the degree of action.

[0106] The pharmaceutical compositions may be in a liquid, aerosol orsolid dosage form, and may be formulated into any suitable formulationincluding, but is not limited to, solutions, suspensions, micelles,emulsions, microemulsions, aerosols, ointments, gels, suppositories,capsules, tablets, and the like, as will be required for the appropriateroute of administration.

[0107] Any suitable route of administration is encompassed by theinvention including, but not being limited to, oral, intravenous,intramuscular, subcutaneous, inhalation, intranasal, topical, rectal orother known routes. In preferred embodiments for the permeabilizationeffect on the CNS, the pharmaceutical composition of the invention isintra-arterially or intra-thecally administered. For use as ananti-cancer medication, the relevant pharmaceutical composition of theinvention is preferably administered orally or intravenously or appliedtopically or by regional perfusion, enema or intra-organ lavage.

[0108] The invention will now be illustrated by the followingnon-limiting examples.

EXAMPLES

[0109] I. Chemical Examples

[0110] For the sake of clarity, procedures for synthesis of particularP-BFA molecules and salts thereof are exemplified below. However itshould be understood that similar procedures are also applicable forsynthesis of other P-BFA molecules of the invention including, but notlimited to, saturated and unsaturated branched chain molecules andcompounds wherein R1 and/or R2 are aliphatic chains comprising a cyclicalkyl group(s). Various pharmaceutically acceptable salts of the P-BFAmolecules could also be obtained including, but not limited to, sodium,potassium, ammonium and alkyl-ammonium salts and salts with divalentcounter-ions.

[0111] All synthesized compounds were characterized by NMR, massspectroscopy and element analyses.

Example 1 Synthesis of Alkyl Phosphates

[0112] Phosphates of the general formula RO—P(O)(OH)₂ were prepared. Rrepresents a branched-chain alkyl moiety of the R₁(R₂)—CH structurewhere R₁ indicates the number of carbons in the side alkyl chain and R₂indicates the number of carbons in the main alkyl chain.

[0113] The synthesis of RO—P(O)(OH)₂ molecules is a three-stageprocedure. In the first stage the corresponding alcohol (R—OH) wasprepared from aldehyde and alkyl bromide using the Grignard reaction(Vogel's, “Textbook of practical organic chemistry”, Wiley, New York,pg. 531, (1996).)

[0114] In the second stage diphenyl phosphate ester was prepared fromthe alcohol and diphenyl phospochloridate:

ROH+ClP(O)(OC₆H₅)₂+C₅H₅N→RO—P(O)(OC₆H₅)₂+C₅H₅N.HCl

[0115] In the third stage alkanyl dihydrogen phosphate was obtained byhydrogenation of the diphenyl ester.

RO—P(O)(OC₆H₅)₂+2H₂→RO—P(O)(OH)₂+2C₆H₆

[0116] 4-Hexadecanyl Diphenyl Phosphate.

[0117] Diphenyl phosphorochloridate (4.0 g, 0.015 mole) was added slowlywhile shaking to a solution of hexadecane-4-ol (2.4 g, 0.01 mole) in drypyridine (5 ml) at room temperature. The flask was stoppered and setaside for 48 hr.; then the contents were poured into ice-cold 1Nhydrochloric acid (100 ml). The heavy oil, which separated was extractedwith ether. The ethereal layer was washed with 1N hydrochloric acid (3times), 5% sodium hydrogen carbonate (5 times), and water (5 times).After being dried (MgSO₄), the ether was removed, and the residue waspurified by column chromatography (Petrol Ether (bp 30-60° C.): Ether,10:1). After evaporation of a solvent 3.5 g of liquid was obtained.Yield 74%.

[0118] 4-Hexadecanyl Phosphate. (3,12-PO₄)

[0119] A suspension of platinum oxide (Adams catalyst) (0.32 g) inglacial acetic acid (20 ml) was shaken under hydrogen atmosphere untilabsorption ceased. The Adams catalyst was then washed well with 2Nhydrochloric acid, water, and finally glacial acetic acid, bydecantation. Solution of 4-Hexadecanyl diphenyl phosphate (3.2 g) inglacial acetic acid (40 ml) was added to the catalyst, and the solutionwas shaken under hydrogen until absorption ceased. The catalyst wasfiltered off and washed with chloroform. The solvents were removed fromthe filtrate in vacuo. The residue was crystallized from petroleum ether(bp 30-60° C.) and dried at 65° C. 2.01 g of final product was obtained.Yield 92%.

[0120] 4-Hexadecanyl Disodium Phosphate. (3,12-PO₄Na₂)

[0121] 4-Hexadecanyl phosphate (1 g, 0.0031 mol) was dissolved inethanol (100 ml). NaOH (0.25 g, 0.0062 mol) was added and the mixturewas stirred for 1 hr. and then evaporated. Ethanol (2×100 ml) was addedand evaporated. Ether (100 ml) was added and evaporated. The residue wascrystallized from acetone (30 ml) and dried at 65° C. (15 mm. Hg). 0.9 gof final product was obtained. Yield 79%.

[0122]¹H-NMR (CD₃OD): δ 0.89 (m, 6H), 1.27 (s, 22H), 1.58 (m, 4H), 4.22(m, 1H). MS (FAB): m/z 367.06 (M+H)⁺.

[0123] 4-Hexadecanyl Monosodium Phosphate. (3,12-HPO₄Na)

[0124] 4-Hexadecanyl phosphate (3.13 g, 0.0097 mol) was dissolved inethanol (150 ml). NaOH (0.37 g, 0.0092 mol) was added and the mixturewas stirred for 48 hrs and then evaporated. Ethanol (2×150 ml) was addedand evaporated. Ether (2×100 ml) was added and evaporated. The obtainedsolid was triturated with acetone (100 ml) and dried under 1 mm. Hgatmosphere overnight. 2.98 g of final product was obtained. Yield 87%.

[0125]¹H-NMR (CD₃OD): δ 0.87 (m, 6H), 1.25 (s, 22H), 1.51 (m, 4H), 4.12(m, 1H). MS (FAB): m/z 345.11 (M+H)⁺.

[0126] 4-Octadecanyl Disodium Phosphate. (3,14-PO₄Na₂)

[0127]¹H-NMR (CD₃OD): δ 0.89 (m, 6H), 1.27 (s, 26H), 1.58 (m, 4H), 4.17(m, 1H). MS (FAB): m/z 395.20 (M+H)⁺.

[0128] 4-Octadecanyl Monosodium Phosphate. (3,14-HPO₄Na)

[0129]¹H-NMR (CD₃OD): δ 0.90 (m, 6H), 1.28 (s, 26H), 1.56 (m, 4H), 4.14(m, 1H). MS (FAB): m/z 373.29 (M+H)⁺.

[0130] 8-Pentadecanyl Disodium Phosphate. (7,7-PO₄Na₂)

[0131]¹H-NMR (CD₃OD): δ 0.90 (m, 6H), 1.30 (s, 20H), 1.60 (m, 4H), 4.12(m, 1H). MS (FAB): m/z 352.93 (M+H)⁺.

[0132] 8-Pentadecanyl Monosodium Phosphate. (7,7-HPO₄Na)

[0133]¹H-NMR (CD₃OD): δ 0.90 (m, 6H), 1.30 (s, 20H), 1.60 (m, 4H), 4.12(m, 1H). MS (FAB): m/z 353.05 (M+Na)⁺.

Example 2 Synthesis of 2-(2′-Propyltetradecanoyloxy)ethyl Phosphate

[0134] This compound was prepared by the same procedures as describedabove in Example 1 using the alcohol: C₃H₇—C(C₁₂H₂₅)H—C(O)—O—CH₂—CH₂—O—.This alcohol was obtained from chloride anhydride of 2-Propyl pentanoicacid and ethylene glycol (Vogel's, “Textbook of practical organicchemistry”, Wiley, New York, pg. 698, (1996)).

[0135] 2-(2′-Propyltetradecanoyloxy)ethyl Disodium Phosphate(3,12-MEG-PO₄Na₂)

[0136]¹H-NMR (CDCI₃): δ 0.87 (m, 6H), 1.24 (s, 22H), 1.54 (m, 4H), 2.33(m, 1H). 3.87 (m, 2H), 4.26 (m, 2H). MS (FAB): m/z 439.19 (M+H)⁺.

[0137] 2-(2′-Propyltetradecanoyloxy)ethyl Monosodium Phosphate(3,12-MEG-HPO₄Na)

[0138]¹H-NMR (CDCI₃): δ 0.87 (t, 6H), 1.24 (s, 20H), 1.42(m, 2H), 1.56(m, 2H), 2.34 (m, 1H). 4.02 (m, 2H), 4.28 (m, 2H). MS (FAB): m/z417.19(M+H)⁺.

Example 3 Synthesis of 2-[2′-(2″-Propyltetradecanoyloxy)-ethoxy]ethylPhosphate

[0139] This compound was prepared by the same procedures as describedabove in Example 1 using the alcohol:C₃H₇—C(C₁₂H₂₅)H—C(O)—O—CH₂—CH₂—O—CH₂—CH₂—O—. This alcohol was obtainedfrom chloride anhydride of 2-Propyl pentanoic acid and diethylene glycol(Vogel's, “Textbook of practical organic chemistry”, Wiley, New York,pg. 698, (1996)).

[0140] 2-[2′-(2″-Propyltetradecanoyloxy)-ethoxy]ethyl Disodium Phosphate(3,12-DEG-PO₄Na₂)

[0141]¹H-NMR (CD₃OD):δ 0.90 (m, 6H), 1.30 (s, 20H), 1.44 (m, 2H), 1.59(m, 2H) 2.39 (m, 1H). 3.72 (m, 4H), 4.09 (m, 2H), 4.24 (m, 2H). MS(FAB): m/z 483.03 (M+H)⁺

[0142] 2-[2′-(2″-Propyltetradecanoyloxy)-ethoxy]ethyl MonosodiumPhosphate (3,12-DEG-HPO₄Na)

[0143]¹H-NMR (CD₃OD):δ 0.90 (m, 6H), 1.30 (s, 20E), 1.44 (m, 2H), 1.59(m, 2H) 2.39 (m, 1H). 3.72 (m, 4H), 4.09 (m, 2H), 4.24 (m, 2H). MS(FAB): m/z 461.31 (M+H)⁺.

[0144] Additional compounds were prepared in an analogous way. ForExample, the compound 2-[2′-(2″-Propyleicosanoyloxy)-ethoxy]ethylPhosphate was prepared by using the alcohol:C₃H₇—C(C₁₈H₃₇)H—C(O)—O—CH₂—CH₂—O—CH₂—CH₂—O—. This alcohol was obtainedfrom chloride anhydride of 2-Propyl eicosanoic acid and diethyleneglycol.

[0145] 2-[2′-(2″-Propyleicosanoyloxy)-ethoxy]ethyl Monosodium Phosphate(3,18-DEG-HPO₄Na)

[0146]¹H-NMR (CD₃OD):δ 0.90 (m, 6H), 1.30 (s, 34H), 1.44 (m, 2H), 1.59(m, 2H) 2.39 (m, 1H). 3.72 (m, 4H), 4.09 (m, 2H), 4.24 (m, 2H). MS(FAB): m/z 544.31 (M+H)⁺.

Example 4 Synthesis of2-{2′-[10″-(Hexadecyl-4-oxy)decyl-1-oxy]ethoxy}ethylphosphate monosodiumsalt (3,12-O—C₁₀-DEG-HPO₄Na)

[0147] This compound was prepared by the same procedures as describedabove in Example 1 using the alcohol:C₃H₇—C(C₁₂H₂₅)H—O—(CH₂)₁₀—O—CH₂CH₂—O—CH₂—CH₂—OH—. This alcohol wasobtained in a two-step procedure: First, reacting 4-Hexadecanol tosylate(Vogel's, “Textbook of practical organic chemistry”, Wiley, New York,pg. 698, (1996)) with monosodium 1,10-decanediole to generate10-(Hexadecyl-4-oxy)decanol. At a second step, the alcohol2′-[10″-(Hexadecyl-4-oxy)decyl-ethyloxyethanol was prepared by reacting10-(Hexadecyl-4-oxy)decanol tosylate (generated as in step 1) withsodium 2-(2-hydroxyethyloxy)ethylate.

[0148]¹H-NMR (CD₃OD):δ 0.90 (m, 6H), 1.30 (s, 38H), 1.44 (m, 2H), 1.59(m, 2H) 3.15 (m, 1H). 3.37 (m, 4H), 3.45-3.62 (6H), 3.95(m, 2H). MS(FAB): m/z 565.45 (M+H)⁺.

Example 5 Synthesis of 2(4-Hexadecanoxy)ethoxyethyl Phosphate

[0149] This compound was prepared by the same procedures as describedabove in Example 1 using the alcohol:C₃H₇—C(C₁₂H₂₅)H—O—CH₂—CH₂—O—CH₂—CH₂—O—. This alcohol was obtained fromsodium 2-(2-hydroxyethyloxy)ethylate and 4-Hexadecanol tosylate.(Vogel's, “Textbook of practical organic chemistry”, Wiley, New York,pg. 698, (1996)).

[0150] 4-Hexadecanesulfonyl chloride.

[0151] 4-Hexadecanol (12.29 g 0.051 mol) and p-Toluenesulfonyl chloride(12 g 0.063 mol) were dissolved Pyridine (100 ml) and stirred overnightat room temperature. Dichloromethane (400 ml) was added. Dichloromethanesolution was then washed well with water, H₂SO₄(3%), water, NaHCO₃ (3%)and water, dried with anhydrous magnesium sulphate (10 g). Afterevaporation of the solvent 20 g of crude 4-Hexadecanesulfonyl chloridewas obtained.

[0152] 2(4-Hexadecanoxy)ethoxyethanesulfonyl Chloride.

[0153] Sodium (3.5 g 0.15 mol) was added to Di(ethylene glycol) (140 ml1.5 mol) at 60° C. in small pieces. To obtained solution 4-Hexadecanoltosylate (crude 20 g) in THF (300 ml) was added at same temperature.Solution was stirred at reflux for 6 hrs. Water (100 ml) was added tothe mixture at room temperature. The mixture was extracted with ethylacetate (300 ml). After evaporation of the solvent the residue waspurified by column chromatography (Petrol Ether (bp 30-60° C.): Ether,1:1). 4 g of 2(4-Hexadecanoyl) ethoxyethyl was obtained. Yield 24%starting from 4-Hexadecanol.

[0154] 2(4-Hexadecanoxy)ethoxyethyl Disodium Phosphate(3,12-ether-DEG-PO₄Na₂)

[0155]¹H-NMR (CD₃OD): δ 0.9 (m, 6H), 1.28 (s, 22H), 1.43 (m, 4H), 3.30(m, 1H), 3.60 (m, 4H), 3.69 (m, 2H), 4.07 (m, 2H). MS (FAB): m/z455.45(M+H)⁺.

Example 6 Synthesis of2-(2′-Propyltetradecanoyloxy)ethoxyethylphosphocholine.

[0156] Phosphocholine of the formulaC₃H₇—C(C₁₂H₂₅)H—C(O)—O—CH₂—C₂H₂—O—CH₂—CH₂—O—PO—(O)—O—CH₂CH₂N⁺(CH₃)₃ wasprepared as follows:

[0157] 2-(2′-Propyltetradecanoyloxy)ethoxyethylphosphocholine(3,12-DEG-PC)

[0158] To a cooled solution (0° C.) of2-(2′-propyltetradecanoyloxy)ethoxyethanol (15.8 g, 0.044 mol) andtriethylamine (10 ml, 0.075 mol) in dry ether (250 ml) was added2-chloro-2-oxo-1,3,2-dioxaphospholane (7 ml, 0.075 mol) in 200 ml of dryether. The mixture was stirred at room temperature for 2 hrs. Thecrystalline (C₂H₅)₃N.HCl that precipitated was filtered off, and thesolvent was removed in vacuum. The residue was dissolved in 500 mlsolution of trimethylamine (0.27M) in anhydrous acetonitrile andtransferred to a pressure bottle. The pressure bottle was kept for 48hrs in an oil bath at 60-65° C. The bottle was then cooled and opened.The solvent was removed, and the residue was purified by columnchromatography (CHCI₃:CH₃OH:H₂O, 1:9:1). The oil obtained afterevaporation of the solvent was lyophilized during 72 hrs at 65° C. 17.4g of slight yellow wax was obtained. Yield 75%.

[0159]¹H-NMR (CD₃OD): δ 0.9 (t, 6H), 1.29:(s, 22H), 1.46 (m, 2H), 1.59(m, 2H), 2.38 (m, 1H), 3.25 (S, 9H), 3.69 (m, 6H), 3.99 (m, 2H), 4.29(m, 4H). MS (FAB): m/z 524.6 (M+H)⁺.

[0160] The following compounds were synthesized by a process analogousto the above-described procedure.

[0161] 2-(2′-Propyleicosanoyloxy)ethoxyethylphosphocholine (3,18DEG-PC)

[0162]¹H-NMR (CD₃OD): δ 0.9 (t, 6H), 1.29 (s, 34H), 1.46 (m, 2H), 1.59(m, 2H), 2.38 (m, 1H), 3.25 (S, 9H), 3.69 (m, 6H), 3.99 (m, 2H), 4.29(m, 4H). MS (FAB): m/z 608.6 (M+H)⁺.

[0163]2-{2′-[10″-(Hexadecyl-4-oxy)decyl-1-oxy]ethoxy}ethylphosphocholine(3,12-O-C₁₀-DEG-PC)

[0164]¹H-NMR (CD₃OD): δ 0.9 (t, 6H), 1.29 (s, 34H), 1.46 (m, 2H), 1.59(m, 2H), 2.38 (m, 1H), 3.25 (S, 9H), 3.69 (m, 6H), 3.99 (m, 2H), 4.29(m, 4H). MS (FAB): m/z 608.6 (M+H)⁺.

Example 7 Synthesis of 2-(2′-Propyltetradecanoyloxy)ethylphosphocholine

[0165] Phosphocholine of the formulaC₃H₇—C(C₁₂H₂₅)H—C(O)—O—CH₂—CH₂—O—PO⁻(O)—O—CH₂CH₂N⁺(CH₃)₃ was prepared bythe same procedure as described above in Example 6, except that thecorresponding alcohol, i.e. 2-(2′-propyltetradecanoyloxy) ethanol, wasused instead of 2-(2′-propyltetradecanoyloxy) ethoxyethanol.

[0166] 2-(2′-Propyltetradecanoyloxy)ethylphosphocholine (3,12-MEG-PC)

[0167]¹H-NMR (CD₃OD): δ 0.9 (t, 6H), 1.29 (s, 22H), 1.46 (m, 2H), 1.59(m, 2H), 2.39 (m, 1H), 3.24 (S, 9H), 3.66 (m, 2H), 4.07 (m, 2H), 4.28(m, 4H). MS (FAB): m/z 480.7 (M+H)⁺.

Example 8 Synthesis of Alkylphosphocholines.

[0168] Phosphocholine compounds of the formula RO—PO⁻(O)—O—CH₂ _(CH)₂N⁺(CH₃)₃ were prepared by the same procedure as described above inExample 6, except that R—OH was used as the alcohol in the initial step.R represents branched chain alkyls of the R₁—C(R₂)H-type.

[0169] 4-Hexadecanyl Phosphocholine (3,12-PC)

[0170]¹H-NMR (CD₃OD): δ 0.91 (t, 6H), 1.28 (s, 22H), 1.57 (m, 4H),3.21(S, 9H), 3.62 (m, 2H), 4.25 (m, 3H). MS (FAB): m/z 408.68 (M+H)⁺.

[0171] 4-Octadecanyl Phosphocholine (3,14-PC)

[0172]¹H-NM. (CD₃OD): δ 0.9 (t, 6H), 1.28 (s, 26H), 1.57 (m, 4H), 3.21(S, 9H), 3.61 (m, 2H), 4.23 (m, 3H). MS (FAB): m/z 436.91 (M+H)⁺.

[0173] 8-Pentadecanyl Phosphocholine (7,7-PC)

[0174]¹H-NMR (CD₃OD): δ 0.92 (t, 6H), 1.32 (s, 20H), 1.59 (m, 4H), 3.23(S, 9H), 3.63 (m, 2H), 4.26 (m, 3H). MS (FAB): m/z 394.35 (M+H)⁺.

Example 9 Synthesis of ω-Branched P-BFA

[0175] ω-branched P-BFA compounds are prepared by the same proceduresdescribed above in Examples 1 and 6 using the relevant ω-alcohols.

[0176] The preparation of ω-branched BFA is a six-stage synthesis. Thestarting reagents are 1-Bromo-ω-alcohol and n-Alkyl-m-alkylketone.

[0177] The first stage is protection of the hydroxyl group of1-Bromo-ω-alcohol by Dihydropyran (DHP) following the procedure asdescribed by Kocienski (“Protecting Group”; Georg. Thieme VerlagStuttgart. N-Y. pg. 83-84 (1994)). Stage 1: Br(CH₂).OH+DHP Br(CH₂).OTHPI II THP denotes a Tetrahydropyranyl ether.

[0178] The second and third stages are standard Grignard synthesis oftertiary alcohol (Vogel's, “Textbook of practical organic chenistry”,Wiley, New York, pgs. 475, 538, (1996)) as follows. Stage 2: mg+Br(CH₂).OTHP BrMg(CH)OTHP Stage 3: BrMg(CH₂).OTHI+RR-CO RR° C.(OH)—(CH₂)00THP IVR, R-denote alkyl groups.

[0179] The fourth stage is reduction of tertiary hydroxy group ofcompound (IV) by ionic hydrogenation reaction following the procedure asdescribed by Carey and Tremper. (JACS, v. 91, p. 2967, (1969)) Stage 4:RR° C.(OH)—(CH₂).OTBP+SiBEt₃ RR!CH(CH₂).OTHP V

[0180] The fifth stage is cleavage of the protection group off theobtained ω-branched alcohol (V) (“Protecting Group”; Georg. ThiemeVerlag Stuttgart. N-Y. pg. 83-84 (1994)).

[0181] The final stage, stage 6, is oxidation of the ω-branched alcoholto the corresponded ω-branched BFA. This was carried out following theprocedure as described by Manger and Lee (Tetraehedron letters, v.22,N.18, p.1655,(1981)).

[0182] Stage 6: RR′CH(CH₂)_(ω)OH+[O]→RR′CH(CH₂)_(ω)COOH

[0183] II. Physico-Chemical Properties:

Example 10 In Vitro Lipophilicity Measurements of DP-BFAs

[0184] The lipophilicity values of various derivatives of branched fattyacids were estimated by comparing the solubility of these compounds inorganic versus aqueous solutions. Octanol and physiological saline wereused, respectively, as the organic and aqueous solutions. The partitioncoefficient (P_(c)) value, i.e. the octanol/saline distribution wasmeasured by the shake-flask technique. The results, i.e. mg/mlsolubility in octanol and water and the calculated LogP_(c), are shownin Table 1. TABLE 1 Octanol-saline partition coefficients (Pa) OctanolWater Molecule* mg/ml mg/ml LogP_(c) 7,7 >50 >25 1.33 3,12 >20 >10 0.993,14 >20 >10 1.26 3,16 >20 9.4 1.26 7,7-PO₄ >10 >10 <5 3,12-PO₄ <5 >2.51.9 3,14-PO₄ >10 <0.5 1.11 3,12-MEG-PO₄ >10 >10 1.09 3,12-DEG-PO₄ >5 81.31 3,12-(ether)-DEG-PO₄ >5 >5 0.83 3,12-PC >10 >10 3,12-MEG-PC >10 >101.22 3,12-DEG-PC >50 >10 0.73

Example 11 Structure-Function Correlations

[0185] In this study a correlation between the physico-chemicalproperties of the branched chain molecules and their biological effectswas assessed. The study was aimed to find out whether there is acorrelation between the chain length of branched chain molecules andtheir potencies in Evans blue (EB) extravasation in rat brain (seeExample 18 for the procedure used in measuring EC₅₀ values). Variousbranched fatty acids of the 3,n-type, wherein one branched chain is of 3carbon atoms and the second hydrocarbon chain, n, is from 7 to 16 carbonatoms, were used. The results are graphically depicted in FIG. 1.

[0186] As can be seen in FIG. 1, BFAs 3,12, 3,14 and 3,16 were found tobe active in permeabilization of the BBB. Under the experimentalconditions employed, the most potent compound among the tested moleculesis BFA-3,12.

[0187] III. Biological Examples:

[0188] III-a) In Vitro Studies

Example 12 Comparative In Vitro Toxicity Study

[0189] Various DP-BFA molecules were screened in cultured Chinesehamster ovary (CHO) AA8 cell line in order to establish their toxicity.LC₅₀ values, i.e. the concentrations causing death in 50% of cellpopulation, were calculated from dose response curves. LC₅₀ values ofphospho-BFAs were compared to those of the corresponding branched fattyacids (=carbo-BFAs).

[0190] Method

[0191] Lactate dehydrogenase (LDH) release was used as an assay for theintegrity of the cells membrane and, thus, for estimation of toxicity.Cells (2×10⁵/ml) were seeded (150 μl/well) in 96 well plates, inRPMI-1640 medium containing 10% FCS. After two days, tested DP-BFAcompounds or the control compound myristic acid were added to the platesin declining concentrations ranging from 500 to 1 μg/ml. Cells wereharvested after one hour. Forty-five minutes before harvesting, 16.5 μlof lysis solution was added to the first 6 wells in order to demonstratecomplete lysis and maximum lactate dehydrogenase release which indicatecell death. Other controls included, two blank wells and five wellscontaining cells with no additional drugs. Following centrifugation(1500 rpm, 5 min), 75 μl of the supernatant was transferred to a newplate with 45 Fl of an LDH assay mix (Tox-7 kit, Sigma). After 10-20minutes at room temperature, absorbance was measured using an ElisaReader 490 nm.

[0192] Toxicity data obtained following 1 hour incubation with differentDP-BFAs is summarized in Table 2. Each experiment was performed intriplicate. TABLE 2 Toxicity of BFAs and their phospho-derivatives oncell cultures Tested compound LC₅₀ (μM) 7,7-Na 65 7,7-PO₄Na₂ 320 3,12-Na165 3,12-PO₄Na₂ 320 3,12-DEG-PO₄Na₂ 320

[0193] In general, the cells were found to be more sensitive tocarbo-BFA and less sensitive to the tested phospo-BFA derivatives.Sub-toxic doses for carbo-BFA were estimated at between 30-100 μMfollowing incubation for 1 hour. Under the same conditions, sub-toxicdoses for the tested phospho-BFA derivatives were estimated to rangefrom 160 μM to more than 640 μM.

[0194] Conclusion: In general, the phospho-BFAs were found to be around2 to 5 times less toxic than the corresponding carbo-BFAs molecules.

Example 13 Effects of DP-BFA on Epithelial Cell Junctions

[0195] Effects of the various BFA derivatives on cell-cell junctionswere investigated using the human adenocarcinoma cell line (A431). Thesecells have a polarized structure characteristic of intestinal cells.

[0196] In this study, quantitative changes in the subcellulardistribution of ZO₂, a protein associated with tight junction complexes(Anderson et al. (1993) Current Opinion in Cell Biology 5: 772-778) wasmonitored.

[0197] A431 cells, cultured in DMEM with serum on glass cover-slips,were incubated for one hour in the presence or absence of sub-toxicdoses of DP-BFA. The cells were fixed and permeabilized for 2 minuteswith a mixture of 3% paraformaldehyde and 0.5% Triton-X-100, and thenfurther fixed for 20 min with 3% paraformaldehyde alone. The fixed cellswere rinsed and incubated for 45 minutes at room temperature with rabbitpolyclonal antibody ZO₂ (Zymed Laboratories, Inc. USA), washed 3 timeswith PBS and incubated for 45 min with fluorescently labelled secondaryantibodies. Stained cover-slips were mounted in Elvanol (Mowiol 4-88,Hoechst, Frankfurt, Germany) before microscopic examination. Thedistribution of the immuno-fluorescently labelled ZO₂ protein wasmonitored.

[0198] A431 cells that were incubated for one hour with pervanadate,which inhibits phosphotyrosine phosphatases and thus disrupts celljunctions, were used as a positive control.

[0199] The fluorescent imaging of ZO₂ indicated that DP-BFA has cleareffects on disruption tight junctions of A431 cells.

Example 14 Effects of DP-BFA on Permeability of Epithelial CellMonolayer

[0200] The aim of this study was to assess the kinetics and overalleffect of the DP-BFA compounds in modifying the permeability of anepithelial cell monolayer, which served as an in vitro model system forbiological barriers and particularly for the intestinal barrier.

[0201] Two approaches were taken in order to determine the effect ofDP-BFA on permeabilization of biological barriers. One approach was tofollow transport of a radioactive labelled compound across theepithelial cell monolayer. A second approach was to monitor changes inelectrical resistance as a measurement indicating the permeability levelof the cell layer.

[0202] Epithelial cells are cultured on a membrane filter (0.4 micron, 1cm²). The membrane with the epithelial cell monolayer is placed betweentwo compartments, a donor and a receiver chamber. The tested P-BFAcompound, at predetermined non-toxic concentration, and ¹⁴C-sucroseradioactive tracer (1 Ci in 0.5 ml buffer) are added to the apical(donor) side. Samples of 0.5 ml each are collected from the basolaeral(receiver) side at 10 minutes intervals for the 90 minutes duration ofthe experiment. Incubation is carried out in tissue culture mediumcontaining 1% FCS while shaking at 70 rpm. The apical chamber andmembrane are transferred into a new receiver chamber at each samplingtime point. This is done in order to keep constant concentration of thetracer in the apical chamber and invariable liquid volumes in bothchambers. The concentration of radioactive tracer in the collectedsamples are estimated using a Trilux microbeta counter (Wallac,Finland). The rate of tracer transport is calculated and expressed asPermeability Coefficient.

[0203] Integrity of the epithelial monolayer used in the experiment ismonitored at the beginning and end of incubation by measuring thetrans-epithelial electrical resistance (TEER) using millicell-ERC(Millipore).

[0204] A parallel set of experiments is conducted using the sameexperimental system as described above but without the addition of theradioactive tracer. Trans-epithelial electrical resistance (TEER) wasmonitored every 10 minutes for a period of 90 minutes.

[0205] Conclusion: the fact that i) P-BFAs significantly increase thepassage of sucrose across the epithelial cell monolayer, and ii)decrease the TEER of the monolayer, support the conclusion that P-BFAcompounds permeabilize biological barrier.

Example 15 Cytotoxic Activity of Various DP-BFAs on Normal and MalignantCell Types

[0206] The cytotoxic activity of various phospho-derivatives ofbranched-chain fatty acids (DP-BFAs) was assayed in vitro in cellcultures that include normal and malignant cell types. The followingcell systems were used:

[0207] Primary fibroblasts (human)

[0208] Normal bone marrow (BM) cells (mouse)

[0209] Primary bronchial epithelial cells (human, from Clonetics, cat.no. CC-2541)

[0210] Caco-2—colon cancer cell line (human, ATTC, HTB-37)

[0211] C6 glioma cell line (rat, ATCC, CRL-2199)

[0212] Neuro2a—neuroblastoma cell line (mouse, ATCC, CCL-131)

[0213] SKBR-3—breast cancer cell line (human, ATTC, HTB-30)

[0214] HL60—myeloid leukemia cell line (human, ATCC, CCL-240)

[0215] U937—myeloid leukemia cell line (human)

[0216] Cells were seeded in a microtiter plate in MEM containing 2 mML-glutamine, 100 units/ml penicillin, 100 ug/ml streptomycin and 10% FCSat 37° C. The cultured cells were incubated, during their linear growthphase in the absence (control group) or presence of various DP-BFAs asindicated. The final concentrations of the tested DP-BFAs ranged from1.5 to 200 microMolars. At the end of the incubation period, which wasfive days for the bone marrow cells and three days for all the othercell lines, the cytotoxic effect of the added P-BFA on the cells wasestimated by using the colorimetric MTT assay. The MTT assay (Mosmann(1983) J. Immunol Methods 65: 55-63) measures mitochondrial reductaseactivity and serves for quantitative assessment of cellular viability.Drug concentration that causes 50% reduction in cell viability incomparison to the control group, is defined as EC₅₀. The EC₅₀ values ofthe tested DP-BFA compounds were calculated from dose response curvesestablished for each of the different assayed cell lines.

[0217] The results are summarized in Table 3. Each EC₅₀ value is anaverage of EC₅₀ values derived from 1-5 independent experiments.

[0218] As can be seen from the results in Table 3, the various DP-BFAsmolecules were active to a different degree in their cytotoxic effect onthe different cells. Among the tested compounds, the most potentcytotoxic agents for malignant cells were 3,12-DEG-PO₄,3,12-(ether)-DEG-PO₄, 3,12-MEG-PC, 3,12-DEG-PC, 3,18-DEG-PC and 3,14-PC.Two of these compounds, 3,12-DEG-PO₄ and 3,12-(ether)-DEG-PO₄, werefound to have the lowest cytotoxic activity on normal epithelial cells.Another compound, 3,12-DEG-PC, was found to have the lowest cytotoxicactivity on normal fibroblasts and normal bone marrow cells. TABLE 3Toxicity of DP-BFAs on various cell lines Normal Normal Normal C6primary bone primary Cell line Caco-2 glioma Neuro2a SKBR HL60 U937fibroblasts marrow epithel Compound EC₅₀ (μM) 3,12-Na 100 76 130 683,12-PO₄-Na₂ >200 105 >200 90 3,12-MEG- >200 60 >200 75 PO₄Na₂ 3,12-DEG-40 38 58 60 25 65 62 >200 PO₄Na₂ 3,12-(ether)- 47 40 37 58 10 65 68 >200DEG-PO₄Na₂ 3,12-PC 150 62 47 3,12-MEG-PC 75 70 50 20 9 160 95 943,12-DEG-PC 113 95 134 40 11 6 170 166 90 3,18-DEG-PC 13 6 80 60 3,14-PC7 4 160 72

[0219] Conclusions: Phospho-BFA compounds have demonstrated cell-typespecific cytotoxic effects. Few of these compounds were shown as mostpotent cytotoxic agents when tested on malignant cell lines, while beingmuch less toxic when tested on normal cells from different tissues.These data suggest that these compounds may be efficient anti-canceragents with low cytotoxic side effects.

Example 16 3,12-DEG-PC Induces Activation of Caspase-3 and DNAFragmentation in a Neuroblastoma Cell Line

[0220] In order to further explore the possible mechanism underlying thecytotoxic effect exerted by the various DP-BFAs, two established markersfor apoptosis, caspase-3 activity and DNA fragmentation (Lincz (1998)Immunol. Cell Biol. 76: 1-19), were studied.

[0221] Neuro2a neuroblastoma cells (ATCC, CCL-131) were seeded in MEMcontaining 2 mM L-glutamine, 100 units/ml penicillin, 100 ug/mlstreptomycin and 10% FCS at 37° C. 24 hours later, when cells were inlogarithmic growth phase, 3,12-DEG-PC was added to a final concentrationof 25 μM, 50 μM or 100 μM. Cells grown in the presence of vehicle onlyserved as control group.

[0222] Assays for caspase-3 activity and DNA-fragmentation wereperformed on cell lysates obtained following, respectively, 6 and 24hours incubation with the drug. Caspase-3 activity was assayed using thefluorogenic substrate Ac-DEVD-AMC (Pharmingen, Becton Dickinson, cat.no. 66081U) and following the manufacture's instructions.DNA-fragmentation was quantified by using the Cell Death Detection Elisaplus kit (Roche, cat. no. 1774 425) and following the manufacture'sinstructions.

[0223] The results are summarized in Table 4. The measured caspaseactivity is expressed as percentage of the activity in the controlplates normalized to protein content. DNA-fragmentation is expressed asOD values at λ=405 nm and is an average of readings from duplicate cellculture lysates. TABLE 4 Apoptosis markers in Neuro2a cells treated with3,12-DEG-PC Caspase DNA activity fragmentation Added drug (% of control)(OD_(405 nm)) None (Control) 100% 0.030 3,12-DEG-PC, 25 μM 210% 0.0363,12-DEG-PC, 50 μM 540% 0.153 3,12-DEG-PC, 100 μM — 0.389

[0224] As can be seen from the results in Table 4, incubation of Neuro2acells with the compound 3,12-DEG-PC, caused about 5-fold increase incaspase-3 activity, and a significant increase in DNA fragmentation.Similar results were obtained when the compound was tested in anothermalignant cell line, caco-2, derived from colon carcinoma.

[0225] Conclusion: Apoptosis may be a possible mechanism for exertingthe cytotoxic effect of phosphocholine derivatives of BFAs in certainmalignant cells.

[0226] III-b) In Vivo Studies

Example 17 In Vivo Model System for Measuring the Effect of DP-BFA onBBB Permeabilization

[0227] Permeabilization of the BBB by DP-BFA was investigated in ratmodel system by monitoring accumulation in the brain of two markers: a)Evans blue dye (EB), which rapidly binds in vivo with albumin (NW 70 kD)and is an indicator for paracellular transport; and b) Fluoresceinsodium salt (F—Na), MW 376 D, which is transported via the cellularspace, and enables monitoring transcellular transport. Upon BBBdisruption the Evans blue-albumin complex irreversibly accumulates inthe intercellular space of the brain. The much smaller tracerfluorescein may be transported intracellularly to occupy both inter andintracellular space in the brain.

[0228] Blood brain barrier permeabilization in Sprague-Dawley rats wasinduced by brief exposure to DP-BFA. The animals were anesthetized withRampun-Imalgen and DP-BFA test compounds were administered via theexternal carotid artery retrograde to the brain (Smith, Q. R. Methods ofstudy. In: Physiology and Pharmacology of the Blood-Brain Barrier. Ed.Bradbury M. W. B. Spinger-Verlag. Berlin-Heidelberg-NY; 1992: 24-52).The pterygo-palathina artery was ligated to avoid escape of infusedsolutions towards the external head vasculature. DP-BFA solutions wereinfused over a period of 30 seconds or half an hour, using a HarvardApparatus Syringe Pump. Blood flow through carotid communis artery wasinterrupted only at the time of infusion.

[0229] All tested DP-BFA molecules were prepared from stock solutions ineither water or ethanol by diluting 200-1000 fold into solution ofisosmotic mannitol (5%) in Tromethamine buffer (Sigma) or PBS (pH 7.4)

[0230] The markers, Evans blue (EB, 25 mg/ml) and fluorescein (F—Na,12.5 mg/ml), in 1 ml solution, were injected intravenously immediatelyfollowing administration of the tested DP-BFA, or after a specific timeinterval if reversibility of action was investigated. Supportive i.v.infusion of F—Na (12.5 mg/ml-100 μl/min) was performed for 8 minutes.Brains were washed with saline solution (60 ml) 10 min after theintracarotid “flash”. Brain ipsilateral and contralateral hemispheresand tumors, where applicable, were homogenized separately with 50%trichloroacetic acid (TCA). The markers concentrations in the cortex andin tumors, where, applicable, were determined spectrofluorometrically(Uyama, O. et al., (1988) J. Cereb. Blood Flow Metab. 8, 282-284:Abraham et al., (1996) Neurosci. Lett. 208, 85-88). The markers contentwas calculated as μg marker per one-gram brain tissue. Standard errorand Student's test for statistical significance were used.

Example 18 Effect of Branched Chain Fatty Acids of Various Chain Lengthson BBB Permeability

[0231] At the first stage of the study, BFA molecules of different chainlengths were screened for their effect in permeabilizing the blood brainbarrier (BBB).

[0232] The experiment was carried out on male Sprague-Dawley ratsweighing 250-320 g. The procedure described above in Example 17 wasfollowed.

[0233] The effect of the tested compounds on the BBB opening wasevaluated by two parameters:

[0234] a) efficacy—The degree of BBB opening estimated in terms ofaccumulation of Evans Blue albumin (μg EB/g brain tissue); and

[0235] b) potency—measured in terms of EC₅₀ and minimal effectiveconcentration (MEC) values. EC₅₀ is the concentration of a testedcompound producing 50% of the maximal effect of Evans Blueextravasation. MEC is defined as the concentration which enablesaccumulation of three times the amount of Evans blue accumulated incontrol animals, in our case a total of 3 μg EB/g brain. The lower theEC₅₀ and MEC values are, the higher the potency of the tested compound.

[0236] The results of evaluation of the effects of different DP-BFAs onBBB permeability are presented in Table 5. TABLE 5 Effects of branchedchain fatty acids on BBB permeability μg EB/g brain Molecule EC₅₀ (μM)MEC (at EC₅₀) 3,7-Na >450 — 0.8 3,10-Na ˜700 — 08 3,12-Na 20 ± 3  6.5 ±0.7 32 ± 4  3,14-Na 40 ± 5  9.1 ± 12  16.5 ± 2.5  3,16-Na 137 ± 17  18 ±2  34 ± 5  7,7-Na 184 ± 21  132 ± 19  4.2 ± 0.6

[0237] As can be seen from the results in Table 5, there is substantialvariation in the degree of Evans Blue extravasation depending on theDP-BFA chain length. Those molecules with the shortest chain length arealmost completely ineffective in this model system. The molecules withthe highest efficacy were BFAs 3,12-Na and 3,16-Na, each elicitsaccumulation of more than 30 μg EB/g brain. In the experimental systemused, 3,12-Na is the most potent permeabilizing agent, having the lowestEC₅₀ and MEC values among the tested compounds.

[0238] Conclusion: Under the conditions of the experimental systememployed, BFA 3/12-Na was found to be the most effective permeabilityenhancer in terms of both potency and efficacy. Therefore, DP-BFAderivatives based on BFA-3,12 were studied in more details.

Example 19 Effect of Different DP-BFAs on BBB Permeability

[0239] The effect of various DP-BFAs on BBB permeability was tested inthe model system of rat brains, as described above in Example 17.Efficacy, namely the level of the BBB opening was expressed as theamount of Evans blue accumulated in the brain (μg EB/g brain). Potencyof the tested molecules was estimated by their calculated EC₅₀ andminimal effective concentration (MEC) values. TABLE 6 Effect ofdifferent DP-BFAs on BBB permeability (in rat brains) μg EB/g brainMolecule EQ₅₀ (μM) MEC (at EC₅₀) 3,12-PO₄-Na₂ 55 ± 1  3.3 ± 0.5 20 ± 3 3,14-HPO₄-Na 67 ± 11 6.2 ± 0.9 10 ± 1  7,7-HPO_(4-Na) 150 — 13,12-MEG-PO₄Na₂ 18 ± 3  11 ± 2  5 ± 1 3,12-DEG-PO₄Na₂ 20 ± 2  3.3 ± 0.518 ± 3  3,12-(ether)-DEG-PO₄Na₂ 54 ± 5  7.3 ± 1.4  26 ± 5.2 3,12-PC 76 ±14 23 ± 4  10 ± 1  3,12-MEG-PC 19.5 ± 1.5  10 ± 1  15 ± 2  3,12-DEG-PC70 ± 12 15 ± 2  14 ± 2  Mannitol  25% — 10 ± 2 

[0240] As can be seen in Table 6, various DP-BFAs molecules promote thepassage of the albumin bound to the Evans Blue tracer across the BBB.The tested compounds differ in their potency and efficacy.

[0241] Under the conditions of this experiment, the most potentderivative of 3,12-BFA was 3,12-DEG-PO₄Na₂ having an ED₅₀ value of 20 μMand minimal effective concentration (MEC) of 3.3 μM. The most effectivecompounds in increasing BBB permeability were 3,12-(ether)-DEG-PO₄Na₂,3,12-PO₄Na₂ and 3,12-DEG-PO₄Na₂. The accumulation levels of Evans Bluedye in rat brain after treatment with EC₅₀ concentrations of the3,12-(ether)-DEG-PO₄Na₂, 3,12-PO₄-Na₂ and 3,12-DEG-PO₄Na₂ compoundswere, respectively, 26, 20 and 18 μg EB/g brain.

Example 20 Effect of DP-BFA on Transport of Evans Blue Bound Albumin andFluorescein Across the BBB

[0242] The effect of various DP-BFAs on BBB permeabilization tomolecules of different sizes was examined in vivo in the model system ofnormal rat brains.

[0243] The transport of two markers was followed: a) Evans blue bound toalbumin (MW 70 kD) as an indicator for paracellular transport; and b)Fluorescein sodium salt (MW 376 D) that represents small size molecules.

[0244] The Evans blue dye (EB) and Fluorescein sodium salt (F—Na) wereinfused into the artery carotis externa over 30 seconds according to theprotocol described above in Example 17. The test compounds were used attheir EC₅₀ concentrations. Control animals were treated with vehiclesolution.

[0245] Evans blue extravasation is expressed as the percentage of thetotal amount of EB accumulated per one gram of brain tissue. Thetransport of fluorescein into the brain, which is dependent on the serumconcentration of this molecule, is presented as percentage of the F—Nalevel in the serum.

[0246] The ratio of accumulation in the brain of F—Na to EB wascalculated and the results are presented in Table 7. TABLE 7 Effect ofdifferent DP-BFAs on BBB permeability to EB-albumin and to Fluorescein %F-Na/ % EB/ Ratio * Molecule g brain g brain F-Na/EB control  0.3 ± 0.080.04 ± 0.01 9.5 ± 1.6 Mannitol- 25% 2.4 ± 0.4  0.6 ± 0.21 4.0 ± 0.53,14-Na 3.8 ± 0.5  0.8 ± 0.07 4.7 ± 1.7 3,12-Na 6.4 ± 1.1 1.8 ± 0.2 4.9± 0.9 3,12-PO₄-Na₂ 8.2 ± 0.9 2.9 ± 0.6 5.9 ± 1.0 3,12-MEG-PO₄-Na₂ 5.0 ±0.8 0.8 ± 0.1 8.4 ± 0.9 3,12-DEG-PO₄-Na₂ 9.0 ± 1.4 1.4 ± 0.2 9.0 ± 1.23,12-(ether)-DEG-PO₄Na₂ 4.4 ± 0.9   4 ± 0.8 1.1 ± 0.2 3,12-PC 3.0 ± 0.50.8 ± 0.1 7.8 ± 1.3 3,12-MEG-PC 5.2 ± 0.8 0.9 ± 0.2 6.9 ± 0.13,12-DEG-PC 2.9 ± 0.5 0.6 ± 0.1 6.8 ± 0.8

[0247] As can be seen in Table 7, normal BBB (=control) is about 10times more permeable to Fluorescein than to EB-albumin complex. Thehyperosmotic permeabilizer, mannitol, increased BBB permeability to EBto a greater extent than to F—Na (F—Na/EB ratio equals 4). Similarly thetested branched fatty acids 3,14-Na and 3,12-Na showed F—Na/EB ratios of4.7 and 4.9, respectively.

[0248] The different phospho-derivatives of BFA affect BBB permeabilityto the two markers in a differential fashion. It was found that 3,12-PO₄increased permeability of the BBB to EB-albumin complex about 1.5 timesmore than the increase in permeability to Fluorescein. On the otherhand, it was found that 3,12-DEG-PO₄ increased permeability of the BBBin a more physiological manner, i.e. increased F—Na transport to thesame extent as EB extravasation. The F—Na/EB ratio in the rat brainsexposed to 3,12-DEG-PO₄ was around 9.

[0249] Conclusions: Various DP-BFA compounds affect BBB permeability indifferent manners. Some DP-BFAs, e.g. 3,12-(ether)-DEG-PO₄Na₂ and3,12-PO₄ (respective F—Na/EB ratios of 1.1 and 5.9) induced selectiveopening of the BBB which favors transport of large molecules overmolecules with low molecular weight. In contrast other molecules, e.g.3,12-DEG-PO₄, equally increased transport of both small molecules suchas F—Na and larger molecules such as EB-albumin.

Example 21 Duration of the Effect of DP-BFA on BBB Opening

[0250] The effects of DP-BFA on Evans Blue and Fluoroscein extravasationin rat brain were examined at various time points following DP-BFAadministration.

[0251] Various DP-BFA compounds were infused, over a period of 30seconds, into the external carotid artery of Sprague Dawley rats,following the procedure described above in Example 17. The testedcompounds were used at their EC₅₀ concentrations. Evans Blue andFluoroscein extravasation in the brain was determined 10, 30, 60, 120and 240 minutes following DP-BFA administration. The content of EvansBlue (μg/g) and Fluoroscein (% of serum) in rat brain was calculatedusing an average of 2-3 individual animals per time point.

[0252] The results obtained in the experimental system as describedabove demonstrate that the DP-BFA effect on the BBB permeabilization isreversible. Moreover, it is suggested that the BBB opening effects ofDP-BFA are relatively short lived. Most tested derivatives ofDP-BFA-3,12 maintain BBB permeability for less than one hour, havingD_(1/2) of around 30 minutes. D_(1/2) is defined as the duration of atleast 50% opening of the BBB using EC₅₀ concentrations of the testedcompounds. Two compounds, 3,12-PC and 3,12-MEG-PC, both comprising aphosphocholine moiety, have shown D_(1/2) which was 4 times longer thanthe other tested 3,12-BFA derivatives, i.e. D_(1/2) of around 120 min.

[0253] Conclusions: DP-BFA was shown to affect BBB opening in the ratbrain in a reversible manner. Various DP-BFA have different durations ofthe permeabilization effect.

Example 22 Toxicology and Safety Studies

[0254] DP-BFA molecules were evaluated for safety and toxicity.Increasing doses of the tested compounds were administeredintra-arterially (i.a.) into rats following the protocol described abovein Example 17. Animal viability was monitored for 24 hours to evaluatelethal concentrations (LC). LC is defined as the minimal concentrationthat caused death. TABLE 8 Comparative safety data of different DP-BFAs(in rats) Safety Molecule* LC (μM) MEC LC/MEC 3,12 40 ± 10 6.5 ± 0.7 6.13,14 78 ± 11 9.1 ± 1.2 8.6 3,16 431 ± 55  18 ± 2  23.9 7,7 360 ± 67  132± 19  2.7 3,12-PO₄ 63 ± 12 3.3 ± 0.5 19.1 3,14-PO₄ 134 ± 18  6.2 ± 0.921.6 3,12-MEG-PO₄ 80 ± 15 11 ± 2  7.5 3,12-DEG-PO₄ 30 ± 5  3.0 ± 0.5 103,12-PC 80 ± 16 23 ± 4  3.5 3,12-MEG-PC 15 ± 2  10 ± 1  1.5 3,12-DEG-PC300 ± 60  15 ± 2  20

[0255] Safety index is defined as the ratio between the lethal dose (LC)and the minimal effective concentration (MEC), wherein MEC correspondsto a calculated concentration of the tested compound that elicitsaccumulation of three times the amount of Evans blue accumulated incontrol animals treated with vehicle only, i.e. total accumulation of 3μg EB. The higher the LC/MEC ratio is, the higher the safety index.

[0256] Conclusions: Introduction of a phosphate group increased safetyindex by 2-3 folds without significantly hampering potency.

Example 23 In Vivo Model System for Measuring the Effect of DP-BFA onBTB Permeabilization (Tumor-Bearing Rats)

[0257] In order to study the ability of DP-BFA compounds to modulateblood tumor barrier (BTB), a model system of C6 glioma-bearing rats, wasemployed.

[0258] Sprague Dawley (SD) rats were inoculated with C6 gliosarcomacells following the procedure described by Bartus et al. (Exp Neurol(1996); 142:14-28). A small burr-hole was made in the frontal scalp boneof the rat fixed in a stereotaxis apparatus. Using a Hamilton syringe,10 μl or 5 μl of media, containing 2.5×10⁵ C6 gliosarcoma cells wereinoculated into the frontal cortex. Coordinates were 1 mm posterior ofbregma, 2.5 mm—lateral, and 3 mm—depth. The needle remained in the placefor 5 min. After removal of the needle, fascia and scalp were sutured.The rats were checked 6-8 days after inoculation, when tumors ofsufficient size (20-60 mg) had developed. The tumor was weighed afterF—Na visualization and dissection. Parts of the rats' brains weresubject to histological examinations and monitored for Evans Blue andFluorescein uptake into ipsilateral tumour tissue, peritumoural tissueand contralateral tissue.

[0259] Results (not shown) obtained from experiments conducted oncontrol tumor-bearing rats (not treated with the compounds of theinvention) demonstrated that permeability of the blood tumor barrier(BTB) for EB and fluorescein is, respectively, about 2-fold and 4-foldhigher than that found for the intact BBB.

Example 24 Effects of Various BFAs Molecules on BBB and BTB Opening(Study in Tumor-Bearing Rats)

[0260] The bilateral tumor model system of C6 glioma-bearing ratsdescribed in Example 23 above was employed for screening of variousderivatives of DP-BFA for their ability to permeabilize the blood brainbarrier (BBB) in comparison to their effect on the blood tumor barrier(BTB). The experimental procedure described in Example 23 was followedexcept that the tumor-bearing rats were further treated with DP-BFA. Thetested compounds, at concentrations around their EC₅₀ values, wereunilaterally infused into the external carotis artery over 30 seconds asdescribed in Example 17. Animals treated with vehicle only serve as acontrol group. Permeabilization effect was quantitated by measuringaccumulation of albumin bound Evans blue dye in tumor (T) versusnon-tumor (NT) tissues.

[0261] Calculated efficacy values and specificity indices are summarizedin Table 9. Specificity index is defined as the ratio between the levelsof EB accumulated in tumor and those accumulated in non-tumor braintissues (T/NT). TABLE 9 Comparison of the BBB and BTB opening by variousDP-BFAs BBB BTB EB μg/g Specificity EC₅₀ * EB μg/g Non-tumor Tumor/Molecule (μM) Tumor brain Non-tumor 3,12-Na 22 ± 4 44 ± 8 43 ± 9  1.1 ±0.3 3,12-PO₄ 10 ± 2 14 ± 6 2.5 ± 1.5 6.3 ± 1.3 3,12-MEG-PO₄ 22 ± 3 25 ±3 21 ± 4  1.2 ± 0.2 3,12-DEG-PO₄  7 ± 2 34 ± 8 2.0 ± 0.2 22 ± 5  3,12-PC52 ± 7 17 ± 2 10 ± 2  1.8 ± 0.5 3,12-MEG-Pc 20 12 6 2 3,12-DEG-PC 60 115 2 Mannitol    25% 12 ± 2 7 ± 2 2.1 ± 0.5

[0262] As can be seen in Table 9, most tested compounds have shownsimilar levels of EB accumulation in tumor and in non-tumor braintissues, i.e. T/NT ratios of about 1 to 2. With mannitol (25%) the EBaccumulation in the tumor was about twice the accumulation in thenon-tumor brain. In the experimental system used, the compound,3,12-DEG-PO₄, showed high specificity for opening the tumor bloodbarrier (BTB) manifested as EB accumulation in tumor which was 22 timeshigher than the EB levels in the non-tumor tissue (T/NT ratio=22).

[0263] Conclusion: 3,12-DEG-PO₄ showed the most specific effect onopening the BTB in C6 glioma tumors. This compound permeabilizes theblood tumor barrier (BTB) of C6 gliomas to a greater extend than thenormal blood brain barrier (BBB). DP-BFA derivatives may, thus, serve asagents capable of specific and selective opening of the BTB.

Example 25 Unilateral DP-BFA Administration in SD Tumor-Bearing Rats

[0264] Evans Blue and Fluoroscein penetration into gliomas tumors wasexamined following unilateral infusion into the external carotis arteryof either 10 μM 3,12-DEG-HPO₄Na (FIG. 2A), 40 μM 3,12-Na BFA (FIG. 2B)or 25% mannitol (FIG. 2C). The relevant compound was infused into ratbrains at 9 to 12 days following inoculation of C6 glioma cells asdescribed in Example 23.

[0265] It may be clearly seen from FIG. 2A Nat the Evans Blue stainingfollowing 3,12-DEG-PO₄ administration to rats with bilateral gliomas isonly in the tumor tissue in the ipsilateral hemisphere. On the otherhand, the carbo counterpart of the DP-BFA, i.e. BFA 3,12, and thehyperosmotic agent, mannitol, equally permeabilized tumor and non-tumorbrain tissues.

Example 26 Effect of DP-BFA on Blood Retinal Barrier (BRB)Permeabilization

[0266] Permeabilization of the blood retinal barrier (BRB) by DP-BFA wasinvestigated in rats by monitoring leakage of the fluorescent marker,fluorescein sodium salt (F—Na), from the blood vessels in the sclera.

[0267] Sprague-Dawley (SD) rats, weighing from 250 to 350 grams, wereanesthetized with a solution of Ketamin (100 mg/ml) and Xylasine (2%)injected at 0.1 ml/100 g body weight. The anesthetized animals wereinjected with 0.25 ml F—Na (12.5 mg/ml) into the jugular vein. Theretinal blood vessels were recorded with a 3-CCD color camera (teliCS5850) attached with a c-mount to the INAMI L-0960 microscope forophthalmic surgery. The microscope is equipped with a light source andan appropriate filter for F—Na detection. After 10 min the retina wascleared from the F—Na. At this point, 1.5 ml of 3,12-DEG-HPO₄Na (12.5μg/ml) was intra-arterially injected over a period of 30 seconds,accompanied with a second i.v. injection of F—Na. Pictures of the bloodvessels were taken at t=0 and 0.25, 0.5, 1, 2, 4, 8 and 10 minutesfollowing administration of the drug. The same experiment was repeatedin a second group of control rats, where 1.5 ml of vehicle (Tab-mannitol5%) was intra arterially injected instead of 3,12-DEG-HPO₄Na.

[0268] By monitoring the distribution of F—Na in the blood vessels atthe posterior part of the animal eye, at the retina level, it wasdemonstrated that by 10 minutes following administration of F—Na alone,all the fluorescent dye was completely cleared from the area surroundingthe retinal blood vessels. However, i.v. injection of F—Na incombination with i.a. injection of 3,12-DEG-HPO₄Na results in F—Naaccumulation around the blood vessels at the retinal level. This leakageof F—Na from the blood vessels, in the presence of 3,12-DEG-HPO₄Na, isan indication for the permeabilization of the blood retinal barrier. Inthe control animals, where only vehicle was administrated instead of theDP-BFA drug, the results were similar to those in the animals injectedwith F—Na alone, namely by 10 minutes following administration, nofluorescence could be detected in the blood vessels or at the areasurrounding them at the retinal level. The results depicted in FIGS.3A-C represent angiographia of the eye, namely pictures of the eye bloodsupply imaging taken at the retinal level as recorded at 30 seconds and10 minutes following administration of F—Na either alone (FIG. 3A), orin combination with 3,12-DEG-HPO₄Na (FIG. 3B) or vehicle (FIG. 3C).

[0269] In accordance with this observation, at 10 minutes following theF—Na administration, higher amounts of F—Na could be detected in thevitreous of the animals treated with 3,12-DEG-HPO₄Na, than in thevitreous of the control animals that were injected with the F—Na andvehicle only (FIGS. 4A-B). The accumulation of the fluorescent signal atthe vitreous is due to the BRB permeabilization and F—Na leakage fromthe blood vessels at the retinal level.

[0270] Conclusion: 3,12-DEG-HPO₄Na enables permeabilization of the bloodretina barrier, and results in F—Na accumulation in the retina andvitreous.

Example 27 Anti-Tumor Activity of DP-BFA

[0271] The anti-tumor effects of various P-BFAs were studied in the invivo model system described above in Example 17. Tested P-BFAs wereintra-arterially administered at the concentrations previouslydetermined as being effective in permeabilization of the BBB and BTB forEvans blue and Fluorescein extravasation. The experimental procedure fortumor inoculation, as described in Example 23, was followed.

[0272] On Day 3-4 following inoculation of the C6 gliosarcoma cells, ratwere cannulated through external carotid artery and the tested P-BFAswere infused over 30 seconds, as described in Example 17, except that inthis experiment no markers were administered. The treated rats weremaintained until Day 7-9, and then sacrificed. Coronal sections wereobtained, stained by eosin-hematoxylin, and reviewed histologically todetermine tumor volume. Animals treated with vehicle only serve ascontrol. Standard error and Student's test for statistical significancewere used. The results are summarized in Table 10.

[0273] As can be seen from the results in Table 10, P-BFA compoundsexhibit cytostatic effect on glioma tumor growth in vivo. Under theexperimental conditions employed, the 3,12-DEG-PO₄ compound exhibitedthe most pronounced anti-tumor effect. Other compounds, e.g.3,12-MEG-PC, also demonstrated a significant anti-tumor activity, thoughto a lesser degree.

[0274] Conclusion: P-BFAs intra-arterially administered into C6glioma-bearing rats exhibit cytotoxic effect on the tumor. TABLE 10Anti-tumor effects of various P-BFAs on tumor growth P-BFA TumorConcentra- volume Molecule tions (μM) (mm³) ± SE P (to control) Control0  8.1 ± 2.22 — 3,12-MEG-HPO₄Na 30 4.2 ± 1.6 >0.1 3,12-PC 50 3.8 ±0.8 >0.1 3,12-MEG-PC 10 2.88 ± 0.6  <0.05* 3,12-DEG-PC 60 3.9 ± 1.0 >0.13,12-DEG-HPO₄Na 5 1.5 ± 0.3 <0.01* 3,12-DEG-PO₄Na₂ 30 1.6 ± 0.4 <0.01*

Example 28 Anti-Tumor Effect of DP-BFA

[0275] The anti-tumor effect of DP-BFA was evaluated by monitoring tumorvolume and appearance in a model system of C6 glioma-bearing rats.Sprague Dawley (SD) rats were inoculated with 2.5×10⁵ C6 glioma cells in5 μl PBS as follows. Rats were mounted into a stereotactic head holderin a flat-skull position. After reflections of the periosteum, a burrhole was preformed with a 1 mm drill in the following coordinates:bregma −1.0, 3.50 mm lateral from the midline on the left side, and at adepth of 3.50 mm. The cell suspension was manually injected at a depthof 3.50 mm using a 10111 Hamilton syringe. The tumor bearing rats weretreated 72 h post inoculation with a single dose, ranging from 5 to 20μM, of 3,12-di-ethylenglycol phosphate (3,12-DEG-PO₄; sodium salt) orvehicle (iso-osmotic buffer, pH-7.4) infused into the external carotidartery for 30 sec. at a velocity of 3 ml/min.

[0276] The pterigo-palatina artery was legated, and the common carotidartery was clamped at the time of infusion.

[0277] Five days after the inoculation of the C6 tumor cells, the ratswere anesthetized with Ketamin/xylazin (1:1) 0.1 ml/100 grams bodyweight, perfused with saline and fixed with 4% Formaldehyde solution.Brains were removed and cryoprotected in a sucrose solution for 48 h.The brains were then sliced in coronal section, stained withHaematoxylin-Eosine and underwent pathology evaluation.

[0278] A significant decrease in the tumor size was observed in thetreated animals in comparison to the non-treated animals. Tumor volumewas 8.1±2.2 mm³ (N=7) in the vehicle-treated animals and 1.6±0.2 mm³(N=12) in the animals treated with 3,12-DEG-PO₄. In addition, themicroenvironment and the nature of the tumor growth were different.Vehicle treated tumor-bearing rats exhibited a remarkable local cellexpansion in the cortex, white matter and meninges. The C6 gliomasappeared irregular and highly infiltrative and showed a high tendency toinvade through the perivascular lymphatic spaces. In contrast, thetreatment with DP-BFA, given once on the 3rd day after cellsinoculation, significantly changed the character of the tumor growth.The C6 glioma invasiveness through the perivascular spaces wasdiminished and a solid tumor with defined border was observed. Somelimited invasion was found into the meninges around the inoculation areaand sub-ependima.

[0279] Conclusion: 3,12-DEG-PO₄ showed a significant anti-tumor effectas inhibiting tumor invasiveness and rate of growth. Thus, DP-BFAcompounds may be useful in inhibiting spreading and invasiveness oftumors.

[0280] The skilled artisan will appreciate that the principles of thepresent disclosure are amenable to many embodiments and variations ormodifications, all of which are within the scope of the invention. Theexamples are intended to be construed as non-limitative, and the scopeof the invention is to be defined by the claims which follow.

1. A compound of the general formula I:

or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 arethe same or different, saturated or unsaturated aliphatic chaincomprising from 2 to 30 carbon atoms; R3 isA-[CH₂]_(m)-B-[CH₂]_(n)-C-[CH₂]_(p)-D, wherein m, n and p are eachindependently zero or an integer from 1 to 12, and A, B, C and D areeach independently selected from a covalent bond, amino, amido, oxygen,thio, carbonyl, carboxyl, oxycarbonyl, thiocarbonyl, phosphate, aminophosphate mono- di- and tri-amino phosphate group with the proviso thatno two oxygen atoms are directly connected to each other; Z₁ and Z₂ arethe same or different, each may be absent or independently selected froma) hydrogen, sodium, lithium, potassium, ammonium, mono-, di-, tri- andtetra-alkylammonium, or b) together with the phospho group form aphospho ester of glycerol choline, ethanolamine, inositol, serine, mono-or oligosaccharide.
 2. The compound according to claim 1, wherein R1 is3 carbon atoms in length and R2 is from 12 to 16 carbon atoms in length.3. The compound according to claim 1, wherein R1 is propyl and R2 isdodecyl.
 4. The compound according to claim 1, wherein R3 comprisesmono-ethylene glycol or di-ethylene glycol moiety.
 5. The compoundaccording to claim 1, wherein the total number of carbon atoms in R2 andR3 together is from 6 to
 26. 6. The compound according to claim 1,wherein the total number of carbon atoms in R2 and R3 together is from16 to
 22. 7. The compound according to claim 1, wherein Z₁ is choline.8. The compound according to claim 1, wherein Z₁ is sodium and Z₂ isselected from hydrogen and sodium.
 9. The compound according to claim 1selected from the group consisting of: 4-Hexadecyl phosphate (3,12-PO₄),4-Octadecyl phosphate (3,14-PO₄), 4-Eicosanyl phosphate (3,16-PO₄),8-Pentadecyl phosphate (7,7-PO₄), 4-Hexadecanoyloxyethyl phosphate(3,12-MEG-PO₄), 2-(4′-Hexadecanoyloxy)ethoxyethyl phosphate(3,12-DEG-PO₄), 4-Hexadecanyloxyethyl phosphate (3,12-(ether)-MEG-PO₄),2-(4′-Hexadecanyloxy)ethoxyethyl phosphate (3,12-(ether)-DEG-PO₄),4-Hexadecyl phosphocholine (3,12-PC), 2-(4-Hexadecanoyloxy)ethylphosphocholine (3,12-MEG-PC), 2-(4-Hexadecanoyloxy)ethoxyethylphosphocholine (3,12-DEG-PC),2-{2′-[10″-(Hexadecyl-4-oxy)decyl-1-oxy]ethoxy}ethylphosphate(3,12-O-C₁₀-DEG-PO₄),2-{2′-[10″-(Hexadecyl-4-oxy)decyl-1-oxy]ethoxy}ethylphosphocholine(3,12-O-C₁₀-DEG-PC), 4-Octadecanoyloxyethyl phosphate (3,14-MEG-PO₄),2-(4′-Octadecanoyloxy)ethoxyethyl phosphate (3,14-DEG-PO₄),4-Octadecanyloxyethyl phosphate (3,14-(ether)-MEG-PO₄),2-(4′-Octadecanyloxy)ethoxyethyl phosphate (3,14-(ether)-DEG-PO₄),4-Octadecyl phosphocholine (3,14-PC), 2-(4-Octadecanoyloxy)ethylphosphocholine (3,14-MEG-PC), 2-(4-Octadecanoyloxy)ethoxyethylphosphocholine (3,14-DEG-PC), 4-Eicosanoyloxyethyl phosphate(3,16-MEG-PO₄), 2-(4′-Eicosanoyloxy)ethoxyethyl phosphate(3,16-DEG-PO₄), 4-Eicosanyloxyethyl phosphate (3,16-(ether)-MEG-PO₄),2-(4′-Eicosanyloxy)ethoxyethyl phosphate (3,16-(ether)-DEG-PO₄),4-Eicosanyl phosphocholine (3,16-PC), 2-(4-Eicosanoyloxy)ethylphosphocholine (3,16-MEG-PC), 2-(4-Eicosanoyloxy)ethoxyethylphosphocholine (3,16-DEG-PC), 10-(4′-Hexadecanoyloxy)decanyl phosphate,10-(8′-Pentadecanoyloxy)decanyl phosphate,2-[2′-(2″-Propyleicosanoyloxy)-ethoxy]ethyl Phosphate (3,18-DEG-PO₄),and 2-(2′-Propyleicosanoyloxy)ethoxy ethylphosphocholine (3,18-DEG-PC).10. The compound according to claim 1 which is2-(4′-Hexadecanoyloxy)ethoxyethyl phosphate, monosodium salt.
 11. Thecompound according to claim 1 which is 2-(4′-Hexadecanoyloxy)ethoxyethylphosphate, disodium salt.
 12. The compound according to claim 1 which is2-(4′-Hexadecanyloxy)ethoxyethyl phosphate.
 13. The compound accordingto claim 1 which is 2-(4-Hexadecanoyloxy)ethyl phosphocholine.
 14. Thecompound according to claim 1 which is 2-(4-Hexadecanoyloxy)ethoxyethylphosphocholine.
 15. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound of the generalformula I or a pharmaceutically acceptable salt thereof,

wherein: R1 and R2 are the same or different, saturated or unsaturatedaliphatic chain comprising from 2 to 30 carbon atoms; R3 isA-[CH₂]_(m)-B-[CH₂]_(n)-C-[CH₂]_(p)-D, wherein m, n and p are eachindependently zero or an integer from 1 to 12, and A, B, C and D areeach independently selected from a covalent bond, amino, amido, oxygen,thio, carbonyl, carboxyl, oxycarbonyl, thiocarbonyl, phosphate, aminophosphate mono- di- and tri-amino phosphate group with the proviso thatno two oxygen atoms are directly connected to each other; Z₁ and Z₂ arethe same or different, each may be absent or independently selected froma) hydrogen, sodium, lithium, potassium, ammonium, mono-, di-, tri- andtetra-alkylammonium, or b) together with the phospho group form aphospho ester of glycerol, choline, ethanolamine, inositol, serine,mono- or oligosaccharide.
 16. The pharmaceutical composition accordingto claim 15, wherein the compound of the general formula I is as definedin any one of claims 2 to
 14. 17. The pharmaceutical compositionaccording to claim 15 further comprising a biologically active agent.18. The pharmaceutical composition according to claim 17, wherein thebiologically active agent is a therapeutic agent.
 19. The pharmaceuticalcomposition according to claim 17, wherein the biologically active agentis an anti-cancer drug.
 20. The pharmaceutical composition according toclaim 17, wherein the biologically active agent is a diagnostic agent.21. Use of a compound of any one of claims 1 to 14 for the manufactureof a medicament.
 22. A method for increasing permeability of abiological barrier comprising exposing said barrier to an effectiveamount of a compound of the general formula I as defined in any one ofclaims 1 to
 14. 23. The method according to claim 22, wherein thebiological barrier is selected from the group consisting of skin,cornea, conjunctival, nasal, bronchial, buccal, vaginal andgastrointestinal epithelium, blood retinal barrier, blood brain barrier,blood testis barrier, blood tumor barrier and blood kidney interphase.24. A method for administration of a biologically active agent into aprivileged site or organ comprising exposing said site or organ to saidbiologically active agent in the presence of an effective amount of apharmaceutical composition of claim 15, thus enabling or increasing thepenetration and/or accumulation of the biologically active agent in theprivileged site or organ.
 25. The method according to claim 24, whereinsaid privileged site or organ is the spinal cord, brain, eye, testis, agland or tumor.
 26. A method for treatment of a central nervous systemdisease or disorder comprising administering to a subject in needthereof an effective amount of a pharmaceutical composition of claim 15in combination with a therapeutic agent.
 27. The method according toclaim 26, wherein said therapeutic agent is selected from the groupconsisting of anti-tumor, anti-viral, anti-microbial, anti-fungal,anti-inflammatory, neuroprotective agents and bioactive peptides andproteins.
 28. A method for treatment of a tumor comprising administeringto a patient in need thereof an effective amount of a pharmaceuticalcomposition of claim
 15. 29. The method according to claim 28 furthercomprising administering to the patient in need thereof an effectiveamount of an anti-cancer drug.
 30. The method according to claim 29,wherein said anti-cancer drug is administered in a separatepharmaceutical composition.
 31. The method according to claim 28,wherein the tumor is in the central nervous system.
 32. The methodaccording to claim 28, wherein the tumor is in the brain.
 33. The methodaccording to claim 28, wherein the tumor is selected from the groupconsisting of carcinoma, glioma, neuroblastoma, retinoblastoma,lymphoma, leukemia, sarcoma and melanoma.
 34. The method according toclaim 28, wherein said tumor is a primary or secondary tumor.
 35. Amethod for treatment of an ophthalmologic disease or disorder comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition of claim
 15. 36. The method according toclaim 35 further comprising administering to the subject in need thereofan effective amount of an additional biologically active agent.
 37. Themethod according to claim 35, wherein the ophthalmologic disease ordisorder is selected from cystoids macular edema (CME), Age-relatedmacular degeneration (ARMD), intraocular infections, intraocularinflammations and intraocular malignancies.
 38. The method according toany one of claims 24 to 37, wherein the pharmaceutical composition isadministered by oral, parenteral or topical administration or byregional perfusion, enema or intra-organ lavage.
 39. The methodaccording to any one of claims 24 to 37, wherein the pharmaceuticalcomposition is intra-arterially administered.
 40. The method accordingto any one of claims 24 to 37, wherein the pharmaceutical composition isintra-thecally administered.
 41. A method for increasing accumulation ofa diagnostic agent in an organ protected by a biological barriercomprising administering to an individual said diagnostic agent incombination with an effective amount of a pharmaceutical composition ofclaim 15, thus increasing accumulation of the diagnostic agent in theorgan protected by the biological barrier.
 42. The method according toclaim 41, wherein said organ protected by a biological barrier is thecentral nervous system.